Clinical Study Secondary Bacterial Infections in Patients with ...downloads.hindawi.com/journals/bmri/2013/376219.pdfbacterial infections in a large group of patients with seasonal

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 376219 6 pageshttpdxdoiorg1011552013376219

Clinical StudySecondary Bacterial Infections in Patients withSeasonal Influenza A and Pandemic H1N1

Karin Liderot1 Marcus Ahl2 and Volkan Oumlzenci1

1 Division of Clinical Microbiology F 72 Karolinska Institutet Karolinska University Hospital Huddinge 141 86 Stockholm Sweden2Department of Infectious Diseases Karolinska University Hospital Huddinge 141 86 Stockholm Sweden

Correspondence should be addressed to Volkan Ozenci volkanozencikarolinskase

Received 2 April 2013 Revised 10 June 2013 Accepted 10 June 2013

Academic Editor Rumiana Koynova

Copyright copy 2013 Karin Liderot et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of the present study is to analyse the secondary bacterial infections in a large group of patients with seasonal influenzaA and influenza A(H1N1) pdm09 Patients diagnosed with seasonal influenza A and influenza A(H1N1) pdm09 between 2005 and2009 were enrolled in the study Data was retrieved from medical records and laboratory information systems (LIS) In total 1094patients with laboratory confirmed influenza were studied There were 352 patients with seasonal influenza A and 742 patientswith influenza A(H1N1) pdm09The patients with influenza A were older and had higher comorbidity than patients with influenzaA(H1N1) pdm09 (119875 lt 0001 and 119875 lt 005 resp) Hospital admission was higher in influenza A group (119875 = 001) In contrastICU admission was higher in patients with influenza A(H1N1) pdm09 than influenza A patients (119875 lt 005) There were highernumbers of bacterial samples taken and culture positivity in patients with influenza A than patients with influenza A(H1N1) pdm09(119875 lt 00001 and 119875 = 001 resp) In both groups the majority of the patients with positive bacterial cultures had underlyingdiseasesThe present study shows that the patient characteristics and the frequency of secondary bacterial infections were differentin patients with seasonal influenza A and in patients with influenza A(H1N1) pdm09

1 IntroductionThe interaction between human influenza viruses with dif-ferent subtypes of human and animal influenza viruses hasbeen shown to give rise to new variants of the virus withpandemic potential [1ndash3] All three previous pandemics 19181957 and 1968 contributed to excess mortality partly due tosecondary bacterial infections [4] In the case of the 1918 pan-demic mortality rates as high as 25 were reported It wassuggested that the secondary bacterial infections were theunderlying reason for these excessive mortality rates [5]After the 1968 pandemic seasonal epidemics of influenzavirus were dominated by AH3N2 virus variants generated byantigenic drift [1 5] and no new pandemics occurred duringthis time However in April 2009 a new influenza AH1N1virus emerged among humans in Mexico and Californiaquickly spreadingworldwide between humans generating thefirst influenza pandemic of the 21st century [6]

During previous influenza pandemics secondary bacte-rial infections caused by Streptococcus pneumoniae Haemo-philus influenzae Staphylococcus aureus and Streptococcus

pyogenes have been important contributors to morbidity andmortality [4 7 8] The information on the epidemiologyof secondary bacterial infections might therefore play asignificant role in reducing mortality and morbidity ratesdue to influenza by early implementation of accurate empiricantibacterial therapy

Previous studies have suggested that influenza A(H1N1)pdm09 differs from the previous influenza pandemics andsecondary bacterial infections seem to play a limited rolein influenza deaths during the current pandemic [9 10]However to our knowledge there is no previous comparativestudy on secondary bacterial infections due to seasonal influ-enza A and influenza A(H1N1) pdm09 These types of com-parative studies might help us to understand the character-istics of the secondary bacterial infections with respectiveinfluenza type

The aim of the present study is to analyse the secondarybacterial infections in a large group of patients with seasonalinfluenza A and influenza A(H1N1) pdm09

2 BioMed Research International

2 Methods

21 Study Material and Design A retrospective study ana-lysing the presence of secondary bacterial infections in pa-tients with either seasonal influenza A (between 2005 and2008) or influenza A(H1N1) pdm09 (2009) was designed Allpatients diagnosed with seasonal influenza A and influenzaA(H1N1) pdm09 between 2005 and 2009 were enrolled inthe study Patients with influenza between 2005 and 2008were grouped as ldquoinfluenza Ardquo It is important to note thatpatients in the ldquoinfluenza Ardquo group were not homogeneoussince several different influenza viruses including H1N1 andH3N2 dominated during different periods of time between2005 and 2008 It was not possible to subgroup these patientsaccording to the type of influenza virus since the moleculartyping of respective virus for each patient was not performedThe patients that had positive bacteriological findings in bothgroups were studied further Reviewers used a standardizedform All data was abstracted frommedical records includingage sex presence of comorbid conditions and clinical pres-entation and course including autopsy reports length ofhospital stay (including intensive care unit stay) and useof antibioticantiviral treatment A positive bacteriologicalfindingwas defined as any growth in blood cultures or growthof relevant pathogenic airway bacteria in airway samplesThemicrobiology results were collected frommedical records andlaboratory information systems (LIS)

22 Detection of Virus Flocked swabs or nasopharyngealaspirates had been used to collect respiratory epithelial cellsfrom the posterior nasopharynx

The laboratory diagnosis of seasonal influenza A wasmade by detection of influenza A-antigen with immunoflu-orescence (IF) Samples were centrifuged at 1000 rpm for10min to pellet the cells for direct immunofluorescence(DFA) The cell pellets were then resuspended in a smallamount of phosphate-buffered saline and 100 120583L was appliedto glass slides by cytocentrifugation (Shandon Cytospin 2Thermo Scientific Waltham MA USA) at 1200 rpm for 10min The slides were air-dried and then fixed in cold acetonefor 10min The cell spots were stained with 25 120583L of con-jugated monoclonal antibodies from a commercial DFA kitfrom PathoDx Respiratory Virus Panel (Diagnostic ProductCorporation Los Angeles CA USA) for 15 minutes at 37∘CEvanrsquos Blue was used as a counterstain After repeated wash-ing in phosphate-buffered saline the slides were mountedin 70 glycerol and examined under microscope A positiveresult was indicated by the presence of at least one intactcell showing specific fluorescence using a fluorescencemicro-scope (Carl Zeiss Oberkochen Germany) The presence ofge50 epithelial cells per glass slide was required for thespecimen to be considered adequate for DFA testing Positiveand negative controls from PathoDx as well as controls fromcultivated influenza A strains were used

For diagnosing pandemic influenza H1N1 real-time-reverse transcriptase-polymerase chain reaction (rRT-PCR)with subtype-specific primers for influenza A(H1N1) pdm09was used A sample volume of 400 120583L was extracted usingthe MagAttract Virus Mini M48 Kit (Qiagen GmbH Hilden

Germany) according to the manufacturerrsquos instructions withthe Qiagen M48 BioRobot The RNA was eluted to a finalvolume of 100 120583LWater was used as a negative control and aninfluenza virus isolate AStockholm22009 H1N1 was usedas a positive control for each extraction The PCR methodused was a one-step rRT-PCR provided by the Swedish Insti-tute for Communicable Disease Control The primers usedwere 51015840 GGC TGC TTT GAA TTT TAC CAC AA 31015840 and 51015840TTT GGG TAG TCA TAA GTC CCA TTT T 31015840 amplifyingthe haemagglutinin gene The probe used was 51015840-FAM-TGCGAT AAC ACG TGC ATGGAAAGTGTC-TAMRA-31015840 Forthe PCR the SuperScript III PlatinumOne-Step QuantitativeRT-PCR systemwas used (Invitrogen Corporation CarlsbadCA USA) The PCR program used was reverse transcriptionfor 15 minutes at 50∘C followed by 2 minutes at 95∘C 45cycles of 95∘C for 5 seconds 60∘C for 60 seconds and 40∘Cfor 30 seconds using LightCycler 480 (Roche DiagnosticsGmbHMannheim Germany) A threshold cycle (119862

119879) ofle40

together with a sigmoid fluorescence curve was needed forthe result to be considered positive

23 Detection of Bacteria All patients with positive sea-sonal influenza A or influenza A(H1N1) pdm09 were cross-examined in the clinical microbiology laboratory database ofKarolinska University Hospital Huddinge for the presenceof positive bacteria cultures from airways or blood Bloodsamples were cultured in the BacTALERT 3D (bioMerieuxInc Durham NC USA) automated blood culture systemDetection of bacteria from clinical samples was made bystandard methods

3 Statistical Analysis

The Fisher exact test and Students t-test were used in cat-egorical comparison and in comparing the two groups re-spectively Values of 119875 lt 005 were considered as statisticallysignificant

31 Ethical Permission The study was approved by the Re-gional Ethical Review Board of Stockholm Sweden (Dnr2010266-31)

4 Results

41 Bacterial Findings In total 1094 patients with laboratoryconfirmed influenza were studied In the study group therewere 352 patients with seasonal influenza A and 742 patientswith influenza A(H1N1) pdm09The numbers of bacteriolog-ical sampling were higher in patients with seasonal influenzaA than patients with influenza A(H1N1) pdm09 240352(6818) versus 99742 (1334) respectively (119875 lt 00001)

The positive bacterial cultures were analysed further inrelation to total numbers of influenza A(H1N1) pdm09 andinfluenza A patients Among the influenza A patients 33352(938) had positive bacteriological samplesThe numbers ofinfluenza A positive patients who had positive cultures fromupper airways lower airways and bloodwere 20352 (568)13352 (369) and 8352 (227) respectively (Table 1)

BioMed Research International 3

Table 1 Numbers of patients with seasonal influenza A and pandemic H1N1 and numbers of relevant bacteria spp isolated from thesepatients Number ( of the total)

Influenza A H1N1 Statistical analysisTotal number of patients 352 742Year 2005ndash2008 2009Patients with bacterial samples taken 240 (6818) 99 (1334) 119875 lt 00001

Patients with positive bacterial findings 33 (938) 38 (512) 119875 = 001

Respiratory culturesPatients with positive upper airway cultureslowast (nasopharynxthroat) 20 (568) 15 (202) 119875 lt 001

S pneumoniae 10 (284) 10 (135) NDH influenzae 5 (142) 4 (054) NDM catarrhalis 8 (227) 3 (040) NDS aureus 0 3 (040) NDS pyogenes (group A streptococci) 2 (057) 0 ND

Patients with positive lower airway cultures (sputumbronchoalveolar lavage) 13 (369) 12 (162) 119875 lt 005

S pneumoniae 5 (142) (01) NDH influenza 2 (057) 0 NDM catarrhalis 1 (028) 1 (013) NDS aureus 3 (085) 5 (067) ND

Coagulase negative staphylococci 0 2 (027) NDS dysgalactiae (group G streptococci) 0 2 (027) NDS pyogenes (group A streptococci) 1 (028) 0 NDEnterobacteriaceae 1 (028) 1 (013) ND

Blood cultureslowastlowast

All positive blood cultures 8 (227) 12 (162) NSS pneumoniae 1 (028) 4 (054) NDS aureus 1 (028) 1 (013) NDH influenzae 1 (028) 0 ND

Viridans streptococci 1 (028) 0 NDS pyogenes (group A streptococci) 0 1 (013) NDCoagulase-negative staphylococci 4 (114) 6 (081) ND

lowastSeveral patients had multiple findings lowastlowastOne patient had multiple findings ND not determined NS not significant

In 38742 (512) of the influenza A(H1N1) pdm09 posi-tive patients the cultures were positive for bacterial growthThe numbers of influenza A(H1N1) pdm09 positive patientswho had positive cultures from upper airways lower airwaysand blood were 15742 (202) 12742 (162) and 12742(162) respectivelyThe different bacterial species identifiedin cultures are depicted in Table 1 There were no cases withmethicillin-resistant Staphylococcus aureus (MRSA) in thestudied material

In total patients with influenza A had higher numbersof positive cultures than patients with influenza A(H1N1)pdm09 (119875 = 001) Numbers of positive upper- and lower-airway cultures were elevated in patients with influenza Athan patients with influenza A(H1N1) pdm09 (119875 lt 001 and119875 lt 005 resp) No difference was observed in the numbersof positive blood cultures between the two groups (Table 1)

42 Patient Characteristics In order to understand the possi-ble underlying mechanisms for the occurrence of secondarybacterial infections the characteristics of patients with pos-itive relevant bacteriological findings in airways and bloodwere analysed in detail The medical records of these patients

were reviewed and the baseline characteristics are depictedin Table 2 For these patients the clinical significance ofthe bacterial findings was determined by assessment of thecliniciansrsquo response to the results from the microbiologylaboratory

There were differences in baseline characteristics betweenpatientswith seasonal influenzaA and patientswith influenzaA(H1N1) pdm09 The median age of patients with seasonalinfluenza A group was significantly higher than influenzaA(H1N1) pdm09 group 575 years versus 305 years respec-tively (119875 le 0001) Women were the majority in both patientgroups 5758 in seasonal influenza A patients and 6579among influenza A(H1N1) pdm09 patients

Total numbers of patients with comorbidity were signif-icantly higher in the seasonal influenza A group (119875 lt 005)When comparing prestudy morbidity in the groups the pa-tients with seasonal influenza A had significantly more car-diovascular disease than the influenza A(H1N1) pdm09 pa-tients 733 (2121) versus 138 (263) respectively (119875 lt005) Two out of 33 (6) seasonal influenza A patients haddiabetes mellitus compared to none among the influenza


Table 2 Baseline characteristics of patients with seasonal influenza A and pandemic H1N1 with relevant positive bacteriological culturesNumber ( of the total)

Influenza A H1N1 Statistical analysisInfluenza patients with bacteriological relevant findings 33 38Agemdashmedian (min-max) 575 (38ndash74) 305 (17ndash43) 119875 lt 0001

Female gendertotal () 1933 (5758) 2538 (6579) NSComorbidityDiabetes mellitus 233 (606) 0 NSChronic lung diseaselowast 533 (1515) 838 (2105) NSChronic cardiovascular disease 733 (2121) 138 (263) 119875 lt 005

Immunosuppressionlowastlowast 1233 (3636) 1138 (2895) NSChronic renal disease 333 (909) 438 (1053) NSTotal number of patients with comorbidity 2633 (7879) 1938 (500) 119875 lt 005

HospitalisationTime between first symptoms to hospital admission (days) 3 4 NDHospital admission 3033 (9090) 2438 (6316) 119875 = 001

ICU admission 233 (606) 1138 (2895) 119875 lt 005

Length of hospital stay including ICU (days) 85 135 119875 = 006

Antimicrobial treatmentAntiviral treatment 1030 (3030) 1838 (4737) NSAntibacterial treatment 2633 (7879) 2538 (6579) NSMortalityOverall mortality 1233 (3636) 338 (789) 119875 = 001

Mortality within 4 weeks of influenza diagnosis 233 (606) 138 (263) NSMortality due to influenza complications 433 (1212) 338 (789) NSND not determined NS not significant lowastasthma or COPD cystic fibrosis lowastlowastkidneyliver transplantation solid tumor chemotherapy acute myelogenousleukaemia (AML) chronic myelogenous leukaemia (CML) autologous stem cell transplant (ASCT) and chronic lymphocytic leukaemia (CLL)

A(H1N1) pdm09 patients Conditions leading to immuno-suppression were equally common in the two groups andincluded lymphoproliferative disease acute myeloid leu-kemia (AML) or chronic lymphocytic leukemia (CLL) stemcell transplantation and secondary immunodeficiency (hy-pogammaglobulinemia or HIV) (data not shown) The pres-ence of chronic lung disease and chronic renal disease wassimilar in both groups

Median time between onset of symptoms and time toseeking medical care was similar in both study groups Incontrast there were considerable differences in length ofhospital stay and intensive care unit (ICU) admission Influ-enza A patients were more often admitted to hospital thaninfluenza A(H1N1) pdm09 patients 3033 (9090) versus2438 (6316) respectively (119875 = 001) Despite high hospitaladmission rates among the influenza A patients the durationof the hospital stay was shorter in the seasonal influenza Apatients compared to influenza A(H1N1) pdm09 patients 85versus 135 days respectively although this difference was notstatistically significant (119875 = 006) Interestingly ICU admit-tance was lower in seasonal influenza A patients compared toinfluenza A(H1N1) pdm09 patients 233 (606) versus 1138(2895) respectively (119875 lt 005)

43 Antimicrobial Treatment Antiviral treatment (Oseltam-ivir or Zanamivir) was used in 1033 (3030) patients withseasonal influenza A and in 1838 (4737) patients with

influenza A(H1N1) pdm09 Treatment time was 5 days forall seasonal influenza A patients and most of the influenzaA(H1N1) pdm09 patients Extended antiviral treatment wasgiven to 6 patients with influenza A(H1N1) pdm09 that hada long ICU stay (gt7 days) Length of antiviral treatment inthese patients ranged between 6 and 35 days (mean= 22 days)All of these patients suffered from preexisting hematologicaldisease (myeloma CLL or AML) or severe chronic lung dis-ease Several of these patients remained PCR positive forinfluenza A(H1N1) pdm09 throughout their ICU stay despiteantiviral treatment

Numbers of seasonal influenza A patients and influenzaA(H1N1) pdm09 patients who received antibacterial treat-ment were 2633 (7879) and 2538 (6579) respectively(Table 2)There was a greater heterogeneity in which antibac-terial regime that was chosen in the seasonal influenza Aepidemics Penicillin and amoxicillin were most commonlyused as oral (PO) therapy closely followed by doxycycline Inmore severe cases when intravenous (IV) administrationwaspreferred cefuroxime alone or penicillin G combined withan aminoglycoside was chosen as empiric therapy When aswitch from IV to PO therapy wasmade amoxicillin or doxy-cycline was used Other empiric antibiotic choices were car-bapenems quinolones and macrolides combined with ami-noglycoside Antibiotic treatments typically lasted for 7ndash10days Patients treated in the ICU for a long time (gt7 days)received IV antimicrobial therapy with a broader spectrum


This was in the majority of cases related to ventilator-associated airway infections or other ICU-related infections(data not shown)

When influenza A(H1N1) pdm09 patients were treatedwith PO antibiotics amoxicillin or amoxicillin-clavulanicacid was preferred as empiric therapy When IV therapywas chosen penicillin G or a secondthird-generation ceph-alosporin was most commonly used Patients admitted to theICU always received a secondthird-generation cephalospo-rin In cases with severe inflammatory response andor severerespiratory failure a quinolone (moxifloxacin) was addedas empiric therapy In cases of septic shockARDS ceph-alosporin therapy was often combined with aminoglycosidesPiperacillintazobactamand carbapenemswere rarely used inthe ICUpatients unless theywere treated in the ICU for a longtime (gt7 days) (data not shown)

5 Discussion

Secondary bacterial infections have been important contribu-tors tomorbidity andmortality during the previous influenzapandemics [4 7 11] It has been shown that S pneumoniaeand S aureus have contributed to excess mortality in thesepatient groups [12] There is no previous study comparingthe characteristics and the occurrence of secondary bacterialinfectionswith different types of influenza viruses in the samesetting

Here we analysed the secondary bacterial infections ina large group of patients with seasonal influenza A andinfluenzaA(H1N1) pdm09 influenza diagnosed atKarolinskaUniversity Hospital during 2005ndash2009

The numbers of influenza A(H1N1) pdm09 positive pa-tients in one year were significantly higher than the numbersof patients with seasonal influenza A in 4 years 742 versus352 patients respectively During 2009 the diagnostic tests inorder to identify influenza A(H1N1) pdm09 were used exten-sivelyThe clinicians were instructed by the Swedish NationalBoard of Health and Welfare to test every suspected caseof influenza A(H1N1) pdm09 influenza to keep track of theextension of the pandemic and to identify the patients in therisk groups that should receive antiviral treatment The highnumbers of influenzaA(H1N1) pdm09 positive patients in thestudy might probably depend on high numbers of samplingfor this virus In the study the diagnostic methods used forseasonal influenza A and influenza A(H1N1) pdm09 werealso different The PCR method which is used for influenzaA(H1N1) pdm09 has previously shown to be more sensitivethan IF which was used for seasonal influenza A between2005 and 2008 [13] The difference in the performance ofthe two methods might also play a role in high numbers ofinfluenza A(H1N1) pdm09 positivity Another possibility isthat the influenza A(H1N1) pdm09 virus infected highernumbers of patients than seasonal influenza A virus

Previous studies have shown that influenza A(H1N1)pdm09 influenza mostly infects young patients [14 15]The present data suggest that the young influenza A(H1N1)pdm09 infected patients who develop severe influenza oftensuffer from preexisting complicating diseases Hospital ad-mission was higher in influenza A group (119875 = 001) In

contrast ICU admissionwas higher in patientswith influenzaA(H1N1) pdm09 than influenza A patients (119875 lt 005) Theinfluenza A(H1N1) pdm09 patients as a group being youngerand healthier were probably more likely to be dischargedfrom the emergency room but the individuals that were hos-pitalized often had a more severe course of infection espe-cially in the subgroup of individuals suffering from immuno-suppression

There were higher numbers of bacterial samples takenand culture positivity in patients with influenza A than pa-tients with influenza A(H1N1) pdm09 (119875 lt 00001 and 119875 =001 resp) However the two patient groups in the studydiffered in size and partly in selection since bacterial cultureswere performed in a larger proportion in the seasonal influ-enza A patients In both groups a majority of the patientswith positive bacterial cultures had underlying diseases Inblood cultures S pneumoniae and coagulase-negative Staph-ylococcus were the most common isolates in both groupsThe threemost common bacterial species isolated from lowerairway samples in influenza A patients were S pneumoniaeS aureus and H influenzae In influenza A(H1N1) pdm09patients lower airway cultures were instead dominated byS aureus Streptococcus dysgalactiae and coagulase-negativestaphylococci The reason for this difference is not known

In the studied material growth of S aureus and S pneu-moniae in lower airways or blood was shown to lead to anti-biotic treatment A majority of positive blood cultures withcoagulase-negative staphylococci were interpreted as con-tamination and subsequently not subjected to antibiotic treat-ment Patients with positive upper airway cultures receivedantibiotic treatment to a lesser extent than those who had apositive lower airway culture and blood culture This couldpartly be explained by increased colonization of upper respi-ratory airways of many bacteria including pathogens duringacute viral respiratory disease as shown by others previously

In the present study 3030 of the patients with seasonalinfluenza A and 4737 of the influenza A(H1N1) pdm09patients received antiviral treatment During the pandemicthe Swedish National Board of Health andWelfare instructedphysicians to initiate antiviral treatment within 2-3 days afterthe beginning of influenza symptoms It has been shown thatpatients with influenza benefit from antiviral treatment if it isinitiated within the first 4 days of illness [16] Several patientswith influenza A(H1N1) pdm09 sought medical help after 4days of flu symptoms and therefore were disqualified forantiviral treatment

Despite a low frequency of secondary bacterial infectionsin the present and previous studies a majority of the patientswith either seasonal influenzaAor influenzaA(H1N1) pdm09received antibacterial treatment Our results concerning bac-terial etiology in these infections suggest that empiric antibi-otic therapy in influenza A and influenza A(H1N1) pdm09patients should be directed primarily against S pneumoniaeand S aureus In the Swedish setting there is in general noneed for empiric coverage of MRSA

In retrospect the course of the influenza A(H1N1) pdm09in Sweden was not as severe as first suspected The lack of agreat number of secondary bacterial infections as presentedhere might be one of the underlying reasons for the benign


course of the disease especially when it is compared with the1918 pandemic [7] However for a few individuals infectionswith the influenza A(H1N1) pdm09 had a severe course dueto viral pneumonia as well as secondary bacterial infectionsTherefore early accurate diagnosis of both influenza aswell assecondary bacterial infections might be important in reduc-ing mortality and morbidity rates in these patients

Authorsrsquo Contribution

Karin Liderot andMarcusAhl have contributed equally to thestudy

Acknowledgments

This study was supported by the grants from the KarolinskaInstitutet No conflict of interests relevant to this paper wasreported by the authors

References

[1] D J Smith A S Lapedes J C De Jong et al ldquoMapping the anti-genic and genetic evolution of influenza virusrdquo Science vol 305no 5682 pp 371ndash376 2004

[2] G J D Smith D Vijaykrishna J Bahl et al ldquoOrigins and evolu-tionary genomics of the 2009 swine-origin H1N1 influenza aepidemicrdquo Nature vol 459 no 7250 pp 1122ndash1125 2009

[3] R J Garten C T Davis C A Russell et al ldquoAntigenic and ge-netic characteristics of swine-origin 2009A(H1N1) influenza vi-ruses circulating in humansrdquo Science vol 325 no 5937 pp 197ndash201 2009

[4] D M Morens J K Taubenberger and A S Fauci ldquoPredom-inant role of bacterial pneumonia as a cause of death in pan-demic influenza implications for pandemic influenza prepared-nessrdquo Journal of Infectious Diseases vol 198 no 7 pp 962ndash9702008

[5] J K Taubenberger and D M Morens ldquo1918 Influenza themother of all pandemicsrdquo Emerging Infectious Diseases vol 12no 1 pp 15ndash22 2006

[6] N M Scalera and S B Mossad ldquoThe first pandemic of the 21stcentury a review of the 2009 pandemic variant influenza A(H1N1) virusrdquo Postgraduate Medicine vol 121 no 5 pp 43ndash472009

[7] J F Brundage and G D Shanks ldquoDeaths from bacterial pneu-monia during 1918-19 influenza pandemicrdquo Emerging InfectiousDiseases vol 14 no 8 pp 1193ndash1199 2008

[8] T R Cate ldquoImpact of influenza and other community-acquiredvirusesrdquo Seminars in Respiratory Infections vol 13 no 1 pp 17ndash23 1998

[9] L Napolitano P Park K Sihler T Papadimos C Chenowethand S Cinti ldquoIntensive-care patientswith severe novel influenzaA (H1N1) virus infection-Michiganrdquo Morbidity and MortalityWeekly Report vol 58 no 27 pp 749ndash752 2009

[10] (CDC) CfDCaP ldquoHospitalized patients with novel influenzaA (H1N1) virus infection-Californiardquo Morbidity and MortalityWeekly Report vol 58 no 19 pp 536ndash541 2009

[11] A L Frank L H Taber and J M Wells ldquoComparison of infec-tion rates and severity of illness for influenza A subtypes H1N1and H3N2rdquo Journal of Infectious Diseases vol 151 no 1 pp 73ndash80 1985

[12] W H Barker and J P Mullooly ldquoPneumonia and influenzadeaths during epidemics Implications for preventionrdquo Archivesof Internal Medicine vol 142 no 1 pp 85ndash89 1982

[13] TGanzenmueller J Kluba BHilfrich et al ldquoComparison of theperformance of direct fluorescent antibody staining a point-of-care rapid antigen test and virus isolation with that of RT-PCRfor the detection of novel 2009 influenza A (H1N1) virus inrespiratory specimensrdquo Journal of Medical Microbiology vol 59no 6 pp 713ndash717 2010

[14] H A Kelly K A Grant S Williams J Fielding and D SmithldquoEpidemiological characteristics of pandemic influenza H1N12009 and seasonal influenza infectionrdquoMedical Journal of Aus-tralia vol 191 no 3 pp 146ndash149 2009

[15] M A Jhung D Swerdlow S J Olsen et al ldquoEpidemiology of2009 pandemic influenza a (H1N1) in theUnited StatesrdquoClinicalInfectious Diseases vol 52 no 1 pp S13ndashS26 2011

[16] N Lee C S Cockram P K S Chan D S C Hui K W Choiand J J Y Sung ldquoAntiviral treatment for patients hospitalizedwith severe influenza infection may affect clinical outcomesrdquoClinical Infectious Diseases vol 46 no 8 pp 1323ndash1324 2008

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2 Methods

21 Study Material and Design A retrospective study ana-lysing the presence of secondary bacterial infections in pa-tients with either seasonal influenza A (between 2005 and2008) or influenza A(H1N1) pdm09 (2009) was designed Allpatients diagnosed with seasonal influenza A and influenzaA(H1N1) pdm09 between 2005 and 2009 were enrolled inthe study Patients with influenza between 2005 and 2008were grouped as ldquoinfluenza Ardquo It is important to note thatpatients in the ldquoinfluenza Ardquo group were not homogeneoussince several different influenza viruses including H1N1 andH3N2 dominated during different periods of time between2005 and 2008 It was not possible to subgroup these patientsaccording to the type of influenza virus since the moleculartyping of respective virus for each patient was not performedThe patients that had positive bacteriological findings in bothgroups were studied further Reviewers used a standardizedform All data was abstracted frommedical records includingage sex presence of comorbid conditions and clinical pres-entation and course including autopsy reports length ofhospital stay (including intensive care unit stay) and useof antibioticantiviral treatment A positive bacteriologicalfindingwas defined as any growth in blood cultures or growthof relevant pathogenic airway bacteria in airway samplesThemicrobiology results were collected frommedical records andlaboratory information systems (LIS)

22 Detection of Virus Flocked swabs or nasopharyngealaspirates had been used to collect respiratory epithelial cellsfrom the posterior nasopharynx

The laboratory diagnosis of seasonal influenza A wasmade by detection of influenza A-antigen with immunoflu-orescence (IF) Samples were centrifuged at 1000 rpm for10min to pellet the cells for direct immunofluorescence(DFA) The cell pellets were then resuspended in a smallamount of phosphate-buffered saline and 100 120583L was appliedto glass slides by cytocentrifugation (Shandon Cytospin 2Thermo Scientific Waltham MA USA) at 1200 rpm for 10min The slides were air-dried and then fixed in cold acetonefor 10min The cell spots were stained with 25 120583L of con-jugated monoclonal antibodies from a commercial DFA kitfrom PathoDx Respiratory Virus Panel (Diagnostic ProductCorporation Los Angeles CA USA) for 15 minutes at 37∘CEvanrsquos Blue was used as a counterstain After repeated wash-ing in phosphate-buffered saline the slides were mountedin 70 glycerol and examined under microscope A positiveresult was indicated by the presence of at least one intactcell showing specific fluorescence using a fluorescencemicro-scope (Carl Zeiss Oberkochen Germany) The presence ofge50 epithelial cells per glass slide was required for thespecimen to be considered adequate for DFA testing Positiveand negative controls from PathoDx as well as controls fromcultivated influenza A strains were used

For diagnosing pandemic influenza H1N1 real-time-reverse transcriptase-polymerase chain reaction (rRT-PCR)with subtype-specific primers for influenza A(H1N1) pdm09was used A sample volume of 400 120583L was extracted usingthe MagAttract Virus Mini M48 Kit (Qiagen GmbH Hilden

Germany) according to the manufacturerrsquos instructions withthe Qiagen M48 BioRobot The RNA was eluted to a finalvolume of 100 120583LWater was used as a negative control and aninfluenza virus isolate AStockholm22009 H1N1 was usedas a positive control for each extraction The PCR methodused was a one-step rRT-PCR provided by the Swedish Insti-tute for Communicable Disease Control The primers usedwere 51015840 GGC TGC TTT GAA TTT TAC CAC AA 31015840 and 51015840TTT GGG TAG TCA TAA GTC CCA TTT T 31015840 amplifyingthe haemagglutinin gene The probe used was 51015840-FAM-TGCGAT AAC ACG TGC ATGGAAAGTGTC-TAMRA-31015840 Forthe PCR the SuperScript III PlatinumOne-Step QuantitativeRT-PCR systemwas used (Invitrogen Corporation CarlsbadCA USA) The PCR program used was reverse transcriptionfor 15 minutes at 50∘C followed by 2 minutes at 95∘C 45cycles of 95∘C for 5 seconds 60∘C for 60 seconds and 40∘Cfor 30 seconds using LightCycler 480 (Roche DiagnosticsGmbHMannheim Germany) A threshold cycle (119862

119879) ofle40

together with a sigmoid fluorescence curve was needed forthe result to be considered positive

23 Detection of Bacteria All patients with positive sea-sonal influenza A or influenza A(H1N1) pdm09 were cross-examined in the clinical microbiology laboratory database ofKarolinska University Hospital Huddinge for the presenceof positive bacteria cultures from airways or blood Bloodsamples were cultured in the BacTALERT 3D (bioMerieuxInc Durham NC USA) automated blood culture systemDetection of bacteria from clinical samples was made bystandard methods

3 Statistical Analysis

The Fisher exact test and Students t-test were used in cat-egorical comparison and in comparing the two groups re-spectively Values of 119875 lt 005 were considered as statisticallysignificant

31 Ethical Permission The study was approved by the Re-gional Ethical Review Board of Stockholm Sweden (Dnr2010266-31)

4 Results

41 Bacterial Findings In total 1094 patients with laboratoryconfirmed influenza were studied In the study group therewere 352 patients with seasonal influenza A and 742 patientswith influenza A(H1N1) pdm09The numbers of bacteriolog-ical sampling were higher in patients with seasonal influenzaA than patients with influenza A(H1N1) pdm09 240352(6818) versus 99742 (1334) respectively (119875 lt 00001)

The positive bacterial cultures were analysed further inrelation to total numbers of influenza A(H1N1) pdm09 andinfluenza A patients Among the influenza A patients 33352(938) had positive bacteriological samplesThe numbers ofinfluenza A positive patients who had positive cultures fromupper airways lower airways and bloodwere 20352 (568)13352 (369) and 8352 (227) respectively (Table 1)


























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Disease Markers



OncologyJournal of





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Journal of

ObesityJournal of



















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Acknowledgments


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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Clinical Study Secondary Bacterial Infections in Patients with ...downloads.hindawi.com/journals/bmri/2013/376219.pdfbacterial infections in a large group of patients with seasonal