emergence of the a / h1n109 influenza virus in 2009 resulted in the first pandemic since 1968 .
the significant role of bacterial coinfection in past pandemics , and in seasonal influenza , has been documented . 
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  during the 19181919 influenza pandemic , the organisms most frequently recovered from the sputum , lung and blood of infected patients were haemophilus influenzae , streptococcus pneumoniae , streptococcus pyogenes and staphylococcus aureus . 

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  in most cases , lung samples taken from patients dying during the 19181919 pandemic demonstrate bacteriologic and histopathologic evidence of severe acute bacterial pneumonia . 
  during the asian and hong kong influenza pandemics of 1957 and 1968 , s.  pneumoniae ,
h.  influenzae and s.  aureus were most frequently isolated from patients with bacterial pneumonia . 

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  in 1957 , s.  aureus and s.  pneumoniae were isolated from 59% and 15% of lung cultures , respectively . 
 
most deaths from s.  aureus were observed in adolescents and young adults . in 1968 , much of the excess mortality was attributed to the increased incidence of bacterial pneumonia : a threefold increase in the incidence of staphylococcal pneumonias and strong correlation between staphylococcal pneumonia and prior influenza infection was observed . 

  during the 2009 influenza pandemic ,
initial clinical descriptions reported a severe respiratory illness with rapid progression to acute respiratory distress syndrome ( ards ) . 
 
codetection of clinically relevant bacteria with influenza a / h1n109 was infrequent , yet 31% of patients admitted to icu had a clinical diagnosis of sepsis and 95% received antibiotics . 
 
bacterial coinfection was documented using histopathological , immunohistochemical and molecular evidence in 22 of 77 ( 286% ) subjects with fatal pandemic influenza a / h1n1 2009 infection .
streptococcus pneumoniae , s.  pyogenes and s.  aureus were the predominant bacterial pathogens detected . 

  despite numerous studies examining coinfection in pandemic influenza a / h1n109 , many questions remain . 

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  many published studies have limited number of critically ill patients enrolled , 
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  have not enrolled all age groups , 
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  have not examined the role of coinfecting respiratory viruses 
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  and have not explored important risk factors such as obesity in those with and without coinfection . 
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  our objectives were ( i ) to determine the burden of bacterial and viral coinfections in adults and children admitted to icus in australia during the first wave of the a / h1n109 pandemic ( 1 june31 august 2009 ) using traditional and molecular techniques and ( ii ) to explore risk factors for and outcomes of those in whom bacteria and other viruses were detected concurrently with a / h1n109 .
all icus contributed data to a multicentre inceptioncohort study involving 187 icus in australia and new zealand . 
 
patient demographics , risk factors , clinical data , complications and outcomes were collected prospectively . 
 
subjects admitted to 14 icus in new south wales and western australia with influenza a confirmed by polymerase chain reaction ( pcr ) were identified .
laboratory results from prior to and following their influenza diagnosis ( 3  days ) were reviewed and collated .
icu admission criteria , diagnostic sampling , antibiotic / antiviral therapy and laboratory processing of respiratory specimens were not standardised across all icus or laboratories .
nucleic acid was extracted from clinical samples using nuclisens easymag ( biomrieux , durham , nc , usa ) . multiplexed tandem polymerase chain reaction ( mtpcr ; ausdiagnostics pty ltd , sydney , nsw , australia ) was performed on all samples to examine for other respiratory viruses including influenza b ( nucleoprotein gene ) , respiratory syncytial virus ( l gene ) , human parainfluenzaviruses 13 ( hemagglutininneuraminidase , nucleoprotein , nucleocapsid gene , respectively ) , adenovirus ( hexon gene ) , metapneumovirus ( nucleoprotein gene ) and picornaviruses ( 5 utr of picornavirus genome ) according to the manufacturer s instructions ( http://www.ausdiagnostics.com ) .
mtpcr was performed on all lower respiratory tract samples for common coinfecting or colonising bacteria including s.  aureus ( nuc , meca , sccmec genes ) , s.  pneumoniae ( lyta , cpsab genes ) , pseudomonas spp .
enterococci and other streptococci / staphylococci ( 16s rrna ) . serial dilution of s.  aureus ( atcc 25923 ) , methicillin resistant s.  aureus ( mrsa ; mrsamws1 ) , s.  pneumoniae ( atcc 49615 ) , pseudomonas aeruginosa ( atcc 27853 ) , escherichia coli ( atcc 25922 ) and escherichia  faecalis ( atcc 29212 ) were prepared , and standard mtpcr curves constructed . 

 
mtpcr was reported positive if the above bacteria were detected as the dominant signal and at a level equal to or greater than that in prepared control samples containing 10  cfu / ml .
controls ( positive and negative ) were included in each mtpcr run . all samples positive by mtpcr
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  subjects were deemed to be assessable for viral coinfection if stored respiratory samples were available for viral mtpcr .
subjects were deemed assessable for bacterial coinfection if ( i ) two or more of the following investigations were performed , ( a ) culture of lower respiratory tract specimen ( bronchoalveolar lavage ( bal)/washing , endotracheal aspirate and sputum ) , ( b ) blood culture and ( c ) urinary antigen detection for s.  pneumoniae and legionella pneumophila or ( ii ) a lower respiratory tract specimen was available for bacterial mtpcr .
communityacquired infection was defined by the onset of symptoms within 48  hours of hospital admission .
the first group included all subjects assessable for both bacterial and viral coinfection ( group 1 ) . to ensure validity of the results ,
a second group was analysed including all subjects in group one and subjects assessable for either bacterial or viral coinfections only ( group 2 ) . to identify the risk factors for and the impact of viral and bacterial coinfection , subjects with and without coinfection
data were analysed using spss version 19.0.0 ( spss inc . , chicago , il , usa ) .
each centre obtained approval from their institutional ethics committee for the original cohort study . 
 
clinical data were available for 198/202 patients admitted to icu with pcrproven influenza a infection .
this included 186 with communityacquired influenza a infection ( 939% ) and 174 ( 879% ) with a / h1n109 ( table  1 ) .
the study population was comparable to the total australian and new zealand icu population diagnosed with influenza during 2009 
  except that seasonal influenza a infection was more common ( 103% versus 43% ; p  <  001 ) and extracorporeal membrane oxygenation was less frequently required ( 42% versus 116% , p  =  0016 ) in the study population . 
demographics , risk factors , clinical features and influenza a subtype of patients including those assessable for coinfection bmi , body mass index ( calculated as weight in kilograms divided by height in metres squared ) .
* includes 93 subjects assessable for both bacterial and viral coinfection , 38 subjects assessable for viral infection only and 45 subjects assessable for bacterial infection only . * * any comorbidity : pregnancy , chronic lung disease , chronic heart disease , diabetes or obesity ( bmi  >  35 ) .
stored upper and/or lower respiratory specimens were available for viral mtpcr in 131/198 ( 662% ) of the study population ( see table  s1a ) .
investigations for bacterial coinfection included blood cultures ( 167/198 : 843% ) , culture of lower respiratory tract specimen ( 115/198 ; 581% ) , urinary antigen detection ( 80/198 ; 404% ) and bacterial culture of pleural fluid , pericardial fluid or lung tissue ( 3/198 ; 15% ) .
lower respiratory tract specimens included bal or washing ( 32/115 ; 278% ) , endotracheal aspirates ( 28/115 ; 243% ) or sputum samples ( 55/115 ; 478% ) .
lower respiratory tract specimens from 34 subjects were available for mtpcr including 21 bronchoscopic specimens , nine endotracheal aspirates and four sputum samples . in total
, 138/198 ( 697% ) of the study population were deemed assessable for bacterial infection ( see table  s1b ) .
ninetythree subjects were assessable for both viral and bacterial coinfections ( group 1 ) , and 176 subjects assessable for any coinfection ( group 2 , table  1 ) . viral coinfection was infrequently observed in both groups : 3/93 ( 32% ) and 6/176 ( 34% ) , respectively ( table  2 ) .
three of the six viral coinfections occurred in children : an infant with influenza a / h1n109 and rsv coinfection , and two children , aged between 3 and 5 , with influenza a and picornavirus coinfections ( one each of influenza a / h1n109 and a / h3 ) .
only one subject had concurrent bacterial and viral coinfection with influenza a : a 50yearold female with concurrent influenza a / h1n109 , adenovirus and s.  aureus infection . 
* one patient had coinfection with influenza a virus , human adenovirus and s.  aureus . 
 
* * two patients had more than one bacterial coinfection identified : ( i ) s.  pneumoniae , p.  aeruginosa and b.  pertussis and ( ii ) s.  pneumoniae and b.  pertussis . 
  using both routine microbiological data and bacterial mtpcr , the rate of bacterial coinfection ranged between 23/93 ( 247% ) and 36/176 ( 205% ) , respectively ( table  2 ) . of patients with bacterial coinfection , 100% ( group 1 ) and 972% ( group 2 )
when only lower respiratory specimens were examined , bacterial coinfection was identified in 4/34 ( 118% ) by mtpcr compared with 2/26 by culture ( 77% ) . 

staphylococcus aureus and s.  pneumoniae were the most frequent pathogens ( table  2 ) .
samples with s.  aureus coinfection ( n  =  17 ) were detected using lower respiratory tract culture ( n  =  14 ; 824% ) , blood cultures ( n  =  3 ; 176% ) , mtpcr ( n  =  2 ; 118% ) and culture of empyema fluid ( n  =  1 ; 59% ) . samples with s.  pneumoniae coinfection ( n  =  10 ) were detected using urinary antigen detection ( n  =  7 ; 70% ) , lower respiratory tract culture ( n  =  1 ; 10% ) , blood cultures ( n  =  1 ; 10% ) and mtpcr ( n  =  2 ; 20% ) . samples with haemophilus coinfection ( n  =  6 ) were detected by using lower respiratory tract culture ( n  =  3 ; 50% ) and blood cultures ( n  =  3 ; 50% ) , other bacterial coinfections ( n  =  6 ) were detected using lower respiratory tract culture ( n  =  2 ; 33% ) , blood culture ( n  =  1 ; 17% ) and mtpcr ( n  =  2 ; 33% ) . when those with and without coinfection were compared , no significant differences were observed in clinical course , influenza type and outcomes .
( group 1 : table  3 ; group 2 : table  s2 ) .
requirement for endotracheal intubation , positive pressure ventilation , vasopressors , extracorporeal membrane oxygenation ( ecmo ) and renal replacement therapy was not significantly different between patients with and without coinfection .
the proportion of patients with influenza a / h1n109 infection was not significantly different between those with and without coinfection ( 20/25 , 800% versus 59/68 , 867% ) . despite a trend towards greater icu mortality in those with coinfection ( 160% versus 59% ) , no significant differences in icu mortality , inhospital mortality , length of icu stay and length of hospital stay
risk factors for and impact of bacterial and viral coinfection in group 1 ( subjects assessable for bacterial and viral coinfection , n  =  93 ) bmi , body mass index ( calculated as weight in kilograms divided by height in metres squared ) . * any comorbidity : pregnancy , chronic lung disease , chronic heart disease , diabetes or obesity ( bmi  >  35 ) . *
* the significance of these variables was maintained if only adults 18  years and older were examined ( p    003 ) : any comorbidity : 7/20 versus 45/68 , p  =  002 ; mean bmi : 2717 versus 3301 , p  =  003 .
significant differences in the frequency of risk factors known to be associated with icu admission were revealed when subjects with and without coinfection were compared ( group 1 : table  3 ; group 2 : table  s2 ) . when subjects in group 1 were compared , subjects with bacterial or viral coinfection were younger [ mean difference in age  =  846  years ( 95% ci  =  0181674  years ) , p  =  0045 ] .
subjects with coinfection were less likely to have significant medical comorbidities compared to those without coinfection ( 320% versus 662% , p  =  0004 , table  3 ) .
when individual comorbidities were examined , those with coinfection were less likely to be obese [ mean difference in body mass index ( bmi ; weight in kilograms divided by height in metres squared )  =  686 ( 95% ci  =  1771196 ) , p  <  001 ] .
a trend towards fewer comorbidities in subjects requiring icu admission with coinfection was observed when compared to those without coinfection : pregnancy , 4% versus 119% ; chronic lung disease , 200% versus 433% ; chronic heart disease , 40% versus 164% ; diabetes , 43% versus 162% ( all not significant ) .
similar differences were observed in subjects assessable for any infection ( group 2 , n  =  176 , table  s2 ) . despite the strong microbiological evidence for bacterial or viral coinfection
, treating physicians most frequently identified the predominant clinical influenza syndrome as viral pneumonitis or ards in coinfected patients ( nine of 25 ; 360% ) . in the remaining subjects ,
the treating physician identified secondary bacterial infection ( 7/25 ; 280% ) , exacerbation of airflow limitation ( 2/25 ; 80% ) and other respiratory syndrome ( 6/25 ; 240% ) as the predominant clinical syndrome .
bacterial and viral coinfection was identified in approximately one in four adults and children admitted to intensive care units in australia with confirmed influenza a infection in 2009 .
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  identified bacterial coinfection in 113/645 adults ( 175% ) admitted with confirmed pandemic influenza a / h1n109 .
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  identified coexisting bacterial pneumonia in 80/325 adults ( 25% ) requiring ventilation with suspected , probable or confirmed pandemic influenza a / h1n109 infection . a similar rate of bacterial coinfection was observed in pandemic influenza a / h1n109 fatalities ( 183290% ) , 
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  significantly more than that observed when all hospitalised children and adults are examined ( bacterial coinfection < 5% ) . 
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  in our group , viral coinfection was infrequent and had little impact on morbidity and mortality .
this is , to our knowledge , the largest study to examine both the burden and impact of respiratory virus coinfection in subjects with severe influenza a infection requiring icu admission during the first wave of the 2009 pandemic .
in contrast , viral coinfection was observed in seven of 39 ( 179% ) patients with severe influenza a / h1n109 infection ( defined as infection resulting in hospitalisation or death ) in argentina by palacios et  al . 

 
rsv was identified in 6/39 of patients with severe disease ( 154% ) compared with 5/160 ( 31% , p  <  001 ) with mild disease .
it is probable that the greater proportion of children and adolescents in the study by palacios et  al .
was reflected in a greater rate of respiratory virus coinfection ( mean age : palacios et  al. 247  years and this study  380  years ) .
variation in the proportion of coinfecting respiratory viruses may also result from seasonal variations in respiratory virus prevalence .
demonstrated that s.  pneumoniae coinfection was an independent predictor of hospital mortality yet this study and that by martinloeches et  al . failed to identify any significant difference between coinfection and icu mortality . 

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  martinloesches et  al . 

  found that coinfected patients were older and had higher apache scores , yet no differences in comorbidities were noted .
our data demonstrate that those with coinfection were younger and had fewer comorbidities normally associated with an increased need for icu admission .
the significance of this finding was preserved if children were included or excluded from the analysis .
martinloesches et  al . demonstrated that subjects with bacterial coinfection were more likely to require invasive ventilation and vasopressors .
this was not demonstrated in this study . despite the failure to identify differences in the severity of illness in subjects with and without coinfection , it is still possible that coinfection was a contributory factor to severe disease .
as enrolment in this study required severe influenza requiring icu admission , it is possible that coinfection was the precipitant that led to icu admission in younger people without comorbidities . to explore this hypothesis further , a clinically wellcharacterised control group not requiring icu admission would be required .
a striking difference is the impact of s.  aureus coinfection in australian icu patients with severe influenza a infection .
staphylococcus aureus was the major pathogen identified in more than 40% of coinfected patients and 96140% of all patients .
all subjects with staphylococcal coinfection in our study had communityacquired infection . similar rates of comorbidities were observed in patients with s.  aureus coinfection compared with patients coinfected with other organisms .
these findings are in contrast to other studies where s.  pneumoniae was the major cause of bacterial coinfection . 
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  despite extensive investigations including pneumococcal urinary antigen testing in addition to respiratory cultures , blood cultures and nucleic acid detection ,
it is , however , possible that this is underestimated because of the frequent use of empiric antibiotics including ceftriaxone ( which has excellent antistreptococcal activity but is associated with increased incidence of s.  aureus 

 ) in patients admitted to icu with communityacquired pneumonia ( cap ) , viral pneumonitis or ards and failure to perform cultureindependent diagnostic tests ( e.g. pneumococcal urinary antigen ) on all patients .
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  these findings have a significant impact on both management and prevention of complications in severe influenza infection . treatment recommendations for adults and children with severe influenza infection include parenteral antibiotics . 

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  the infectious diseases society of america recommends a thirdgeneration cephalosporin and a macrolide / fluoroquinolone in critically unwell adults with influenza a / h1n109 influenza . 
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provision of cover against mrsa is recommended in those admitted to icu , with necrotising / cavitatory infiltrates , empyema or microscopy demonstrating grampositive cocci in clusters in a respiratory specimen . 
 
the british thoracic society recommends a betalactamase stable penicillin or second/thirdgeneration cephalosporin together with a macrolide in adults with severe influenzarelated pneumonia .
empiric cover against mrsa is recommended in subjects hospitalised within the last few months or those not responding to empirical therapy . 
 
these data support the use of empiric antibiotics with activity against locally circulating clones of s.  aureus in all patients admitted to icu with severe influenza infection .
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  only three patients diagnosed with invasive pneumococcal disease would have been recommended to receive pneumococcal vaccination using current australian guidelines . 
 
our data suggest that the impact of immunising young children , the elderly and those with underlying medical conditions would have little impact on morbidity and mortality in australians with severe influenza infection .
the present study has several potential limitations . despite a uniform approach to diagnosis of cap
the observational nature of this study means that the diagnostic approach varied between different patients and icus .
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  adequate lower respiratory specimens and samples for cultureindependent laboratory tests ( e.g. pneumococcal urinary antigen ) were not routinely submitted for microbiological analysis .
in addition , it is likely that many subjects were administered antibiotics prior to specimen collection .
it is therefore possible that the true burden of bacterial and viral coinfection is underestimated . despite the use of quantitative and/or semiquantitative laboratory methods , coinfecting and
we have attempted to overcome this by further analysing lower respiratory tract specimens using quantitative nucleic acid amplification techniques , a test not routinely used for the diagnosis of cap .
this approach was hampered by limited lower respiratory sampling in the 14 icus enrolling subjects .
it is possible , however , that the detection of bacteria colonising the lower respiratory tract at high concentrations may falsely elevate the observed rate of bacterial pneumonia .
furthermore , viral coinfection may have been underestimated as emerging viral pathogens , such as human bocavirus and coronaviruses , were not tested for .
additional pathogens may have been detected if upper and lower respiratory samples were available for analysis on all patients . 
 
it is probable that the burden of coinfection would be higher in subjects with radiologically confirmed pneumonia and those examined prior to receipt of antibiotics .
the burden of bacterial and viral coinfection in 198 adults and children admitted to icu during the first wave of the influenza a / h1n109 pandemic was estimated to be 233269% .
coinfection had little impact on severity of illness or outcome in those admitted to intensive care but may have contributed to the need for icu admission in those without other risk factors .
empiric parenteral antibiotics with antistaphylococcal activity should be strongly considered in those with severe influenza infection . to estimate the true burden and impact of bacterial and viral coinfection , a prospective study with uniform sampling using both culture and molecular assays
further development of new diagnostic methods is urgently required , particularly to assist with diagnosing bacterial pneumonia .
funding for the study was provided from a untied grant through the investigatorinitiated grants program of merck us  grant # iisp3732 .
ccb , sarw , ded , is and jri were involved in the design of the study .
sarw , is and jri were involved in the nested cohort study ( webb et  al . , nejm 2009 ) .
ccb , jk , sjvh , hf , amk were involved in the processing and storage of laboratory specimens .
ccb , jk and ang were involved in additional microbiological processing . statistical analysis and the original draft

 table s1 . differences between subjects assessable and not assessable for ( a ) viral coinfection and ( b ) bacterial coinfection . 

. risk factors for and impact of 
 bacterial and viral coinfection in group 2 ( subjects assessable 
 for any coinfection , n  =  176 ) .
 
 please cite this paper as : blyth et  al . 
 ( 2013 ) the impact of bacterial and viral coinfection in severe influenza . influenza and other respiratory viruses 7(2 ) 168176 . 
 
 background   many questions remain concerning the burden , risk factors and impact of bacterial and viral coinfection in patients with pandemic influenza admitted to the intensive care unit ( icu ) . 
 
 objectives   to examine the burden , risk factors and impact of bacterial and viral coinfection in australian patients with severe influenza . 
 
 patients / methods   a cohort study conducted in 14 icus was performed . 
 patients with proven influenza a during the 2009 influenza season were eligible for inclusion . 
 demographics , risk factors , clinical data , microbiological data , complications and outcomes were collected . 
 polymerase chain reaction for additional bacterial and viral respiratory pathogens was performed on stored respiratory samples . 
 
 results   coinfection was identified in 233269% of patients with severe influenza a infection : viral coinfection , 3234% and bacterial coinfection , 205247% . 
 staphylococcus aureus was the most frequent bacterial coinfection followed by streptococcus pneumoniae and haemophilus influenzae . 
 patients with coinfection were younger [ mean difference in age  =  846  years ( 95% ci : 0181674  years ) ] , less likely to have significant comorbidities ( 320% versus 662% , p  =  0004 ) and less frequently obese [ mean difference in body mass index  =  686 ( 95% ci : 1771196 ) ] compared to those without coinfection . 
 
 conclusions   bacterial or viral coinfection complicated one in four patients admitted to icu with severe influenza a infection . despite the coinfected patients being younger and with fewer comorbidities , no significant difference in outcomes was observed . 
 it is likely that coinfection contributed to a need for icu admission in those without other risk factors for severe influenza disease . 
 empiric antibiotics with staphylococcal activity should be strongly considered in all patients with severe influenza a infection . 
Introduction
Patients and methods
Results
Discussion
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Author contributions
Supporting information