Abstract
The emergence of new influenza virus subtypes has rekindled the interest in the clinical course and outcome of patients with influenza-associated pneumonia.
Based on prospective data from 5,032 patients with community-acquired pneumonia (CAP) included in the German Competence Network for Community-Acquired Pneumonia (CAPNETZ), we studied the incidence, clinical characteristics and outcome of patients with influenza-associated CAP and compared these findings with patients without influenza. Diagnosis relied on a positive PCR for influenza in throat washings.
160 patients with influenza-associated CAP were identified (3.2% of total population, 12% of those with defined aetiology). 34 (21%) patients with seasonal influenza had a concomitant pathogen (mostly Streptococcus pneumoniae). Patients with influenza-associated CAP were significantly older, had been vaccinated less often and had preceding antibacterial treatment less often. 30-day mortality was low (4.4%) and not different to that of patients with pneumonia caused by bacterial (6.2%) or viral (other than influenza) pathogens (4%). Patients with influenza plus a bacterial pathogen (mixed influenza-associated pneumonia) had a higher mortality than those with pure influenza-associated pneumonia (9% versus 3.2%).
Mortality was higher in patients with mixed compared with pure influenza-associated pneumonia. However, we could not observe any excess mortality in patients with influenza-associated pneumonia.
Influenza infection is generally assumed to be responsible for a considerable excess mortality, particularly in elderly and comorbid patients, at least in seasons with high influenza activity 1. Most recently, from 1976–1977 through to 2002–2003 seasons, an annual average of >25,000 influenza-associated respiratory and circulatory deaths (9.9 deaths per 100,000) have been calculated in the USA 2. Similarly, average influenza-associated excess mortality in Germany is estimated to be around 16 per 100,000 inhabitants 3. Corresponding vaccination policies are based on this perception, and in fact, several studies have shown considerable decreases in influenza mortality in vaccinated subjects 1, particularly in high-risk patients 4. Moreover, the Infectious Disease Society of America/American Thoracic Society guidelines recommend specific anti-viral treatment in patients with evidence for influenza aetiology in community-acquired pneumonia (CAP) 5. Together with allusions to the Spanish flu disaster, influenza might be considered as a major killer in respiratory tract infections.
In sharp contrast to this, there are only very few reports addressing influenza in CAP and reported mortality rates do not seem to support excess mortality in the presence of influenza-associated pneumonia 6–8. It has to be realised that the notion of influenza causing excess mortality is based on surveillance data and statistical models to estimate the burden of disease 9. However, these data only show associations and not causal relations of excess mortality. In fact, excess mortality might be caused by reasons other than respiratory diseases.
Therefore, we studied the incidence, initial severity, clinical presentation and outcomes of patients with influenza-associated pneumonia within the large German Competence Network for Community-Acquired Pneumonia (CAPNETZ) cohort in order to assess the contribution of influenza-associated pneumonia to pneumonia mortality. We compared this data with the general population without influenza-associated pneumonia. We also investigated the bearing of secondary bacterial infection.
MATERIALS AND METHODS
Patient population
A detailed description of the CAPNETZ methodology is given elsewhere 10. The study was approved by the ethical review board and all patients included gave informed consent.
Data collection
In this prospective study, all demographic, clinical and diagnostic data of the patients were recorded using standardised web-based data sheets created by 2mt® (Ulm, Germany). The study period comprised 58 months starting on June 1, 2002 and ending April 30, 2007, thus including five autumn–winter seasons.
Microbiological and virological processing and examination
Methods applied were as described previously 10, 11. In short, sputum and/or other respiratory secretions were immediately processed in the participating local microbiological laboratories according to the German Quality Standards in Clinical Microbiology and Infectious Diseases (MIQ).
All respiratory specimens and blood cultures if available were immediately processed in the local microbiology laboratories of the participating clinical centres. Gram-staining and culture were performed for all respiratory samples. Validated sputum, blood culture samples, pleural fluid and undiluted and serially diluted tracheobronchial aspirates, PBS and bronchoalveolar lavage fluid (BALF) samples were plated on blood-sheep agar, CDC agar and chocolate agar. Undiluted PBS and BALF samples were also cultured on charcoal–yeast extract agar if Legionella spp. was suspected. Urine was tested for the presence of Streptococcus pneumoniae and Legionella spp. antigen. Standardised throat washings of all patients using sterile 0.9% NaCl were sent immediately to the German reference centre for influenza (Berlin, Germany). PCR for influenza A and B was performed in the throat washings of all 5,032 patients.
RNA extraction and cDNA synthesis
Viral RNA was extracted using a commercial kit (QIAamp Viral RNA Kit; Qiagen, Hilden, Germany). Briefly, 150 μL of clinical specimen (throat swab, nasal swab or gargle) were mixed with an equal volume of lysis buffer AL, heated for 15 min at 70°C and applied to a spin column. Unbound material was removed by several washing steps and the RNA eluted using 50 μL of RNAse-free water. The complementary DNA (cDNA) synthesis was carried out at 37°C for 1 h using 10 μL of RNA, 100 U of murine leukaemia virus reverse transcriptase (Gibco BRL, Life Technologies GmbH, Karlsruhe, Germany), 10 mM dithiothreitol, 150 μM (each) dATP, dCTP, dGTP and dTTP (20 U RNAsin (Promega, Mannheim, Germany)) and 0.25 μM random hexamer primers.
PCR and sequence analysis
The TaqMan-PCR was carried out in a 96-well flat-bottomed microtitre plate format (PerkinElmer, Rodgau, Germany). The PCR mix was made up to a volume of 25 μL, containing 5 μL of cDNA, 50 mM Tris-hydrochloride, pH 9, 50 mM KCl, 4 mM MgCl2, 0.2 mM (each) dATP, dCTP, dGTP dUTP, 0.5 units uracil-N-glycosylase (Gibco BRL, Life Technologies), 1.25 units Taq DNA polymerase (InViTek, Berlin, Germany), 0.25 μM each of the forward and reverse primer, 0.2 μM of a fluorescence-labelled probe and 1 μM ROX as passive reference. Virus identification and further subtyping was carried out as described previously 11 with some modifications (primer and probe sequences available on request). The cDNA was amplified by 45 two-step cycles (1 min 92°C and 1 min 60°C). The amplification in the TaqMan-PCR was followed on the ABI Prism™ 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA). The plate was scanned at 518 nm (carboxyfluorescein) and 582 nm (carboxytetramethylrhodamine). Data acquisition analysis was handled by using the Fluorescence Data Manager (Perkin Elmer) and Excel (Microsoft Corporation, Redmond, WA, USA) spreadsheets. ROX was used as a passive reference to which the reporter dye signal was normalised (Rn) during data analysis.
Definitions
We defined patients with PCR-positive influenza respiratory samples as “influenza-associated pneumonia” as we ignore the exact contribution of influenza virus infection as an aetiological pathogen in CAP. In particular, we cannot exclude the presence of bacterial co-pathogens that we might have missed.
Comparisons
We compared clinical characteristics, severity at admission and outcomes of: 1) patients with influenza-associated pneumonia and those without; and 2) pure influenza-associated pneumonia and influenza pneumonia with bacterial co-infection (mixed influenza-associated pneumonia).
Statistical analysis
Comparisons between groups were performed by means of the Chi-squared test for categorical variables or Fisher’s exact test in the case of small expected frequencies and ANOVA for continuous variables including multiple comparisons. All analyses were performed with SPSS software (SPSS 10.0; SPSS, Inc., Chicago, IL, USA). All tests of significance were two-tailed and α was set at 0.05.
RESULTS
General characteristics of study population
Overall, 5,032 patients with CAP from 12 clinical centres throughout Germany were included in our analysis from 2002 to 2007. The 2,781 male and 2,251 female patients had a mean age of 60±18 yrs. 65% (n = 3,274) of the patients were hospitalised when first contacted for participation in CAPNETZ. 307 (6%) patients were nursing home residents. 31% of the patients were smokers. Severity scores as assessed using the confusion, respiratory frequency and blood pressure in those aged ≥65 yrs (CRB-65) index were available for 90% of the patients and distributed as follows: CRB-65 0 (37%), 1 (38%), 2 (13%), 3 (3%) and 4 (0.3%), respectively. CRB-65 status was not available for 8.7% of the patients. 134 (3%) patients required mechanical ventilation. 238 (4.7%) patients died within 30 days after diagnosis and 180-day mortality in all patients was 8.8% (445 patients).
General microbial patterns
In 1,337 (27%) patients a definite pathogen causing CAP could be identified. 124 (2.5%) patients carried more than one pathogen.
S. pneumoniae was identified as the predominant respiratory pathogen in the study population, followed by Mycoplasma pneumoniae. In <5% of the patients Legionella spp., Haemophilus influenzae and Staphylococcus aureus were identified. Even less frequent were Moraxella catarrhalis and Chlamydophila pneumoniae.
Overall, 160 patients (3.2% of total population, 12% of those with defined aetiology) had CAP associated with seasonal influenza A (134 patients) and B (26 patients), respectively. Incidence was higher in autumn–winter seasons (4.7% and 16.2%, respectively).
124 of these patients were classified as pure influenza-associated CAP, 34 (21%) patients had influenza and a concomitant pathogen (mixed influenza-associated pneumonia), predominantly S. pneumoniae (n = 17) (table 1).
In an additional 73 patients, respiratory syncytial virus (RSV) (29 patients), enterovirus (28 patients) and adenovirus (16 patients) were detected.
Ambulatory and hospitalised patients
3,274 patients were hospitalised; 102 (3%) of these had influenza-associated pneumonia. 78 (62%) of the patients with pure influenza-associated pneumonia and 24 (71%) of the patients with mixed influenza-associated pneumonia had been admitted to a hospital. Data concerning intensive care unit (ICU) admission of the patients were scarce and thus not included in the analysis.
Seasonal variability of seasonal influenza
Seasonal variability is reflected in figure 1. As expected, there were seasonal peaks of incidence during January and March each year, with considerable variations in numbers within years. In accordance with national surveillance data, 2003, 2005 and 2007 were years with high influenza activity, whereas 2004 and 2006 were relatively modest 12.
Seasonal distribution of influenza-associated pneumonia throughout five autumn–winter seasons. ♦: number of cases; ▪: number of deaths.
Vaccination status
26% of patients with influenza-associated pneumonia had received seasonal influenza vaccination and 9% pneumococcal vaccination. These rates were not significantly different from the comparator groups.
Clinical characteristics of influenza-associated CAP
Patients with pure influenza-associated pneumonia were significantly older, had received significantly less preceding antibiotic therapies and had significantly lower leukocyte counts. They had been less frequently vaccinated (26% versus 33%) (table 2).
Patients with mixed viral pneumonia had no distinguishing features when compared with the study population without influenza. If comparing patients with pure influenza-associated pneumonia to patients with bacterial co-pathogens, patients with bacterial co-pathogens were significantly younger and had significantly higher inflammatory markers (table 2).
Outcome of influenza-associated CAP
Anti-viral therapy was not a specific query on the data sheet but could be documented voluntarily. Thus, no data regarding anti-viral treatment were analysed.
Nine patients had a high CRB-65 score of 3 (seven patients) or 4 (two patients) when admitted and four patients received mechanical ventilation.
Seven patients with influenza-associated pneumonia died (4.2% of patients with influenza). Of these, three had a concomitant pathogen, including S. pneumoniae (n = 2) and H. influenzae (n = 1). Mortality was three out of 34 (9%) if a co-pathogen was present, as compared to four out of 126 (3.2%) in the case of influenza being the only pathogen (p = 0.166). The clinical characteristics of these patients are listed in table 3. Lethal outcome was associated with pneumonia severity at admission (CRB-65 p< 0.0001), smoking habits (p = 0.02) and older age (p = 0.068).
DISCUSSION
The main findings of this study are the following: 1) influenza was an important pathogen associated with CAP in our population, with an incidence of 160 cases (3.2% of the total population; 12% of patients with defined aetiology); 2) related to autumn–winter seasons, the incidence was even higher (4.7% of the total population and 16.2% of patients with defined aetiology); 3) influenza-associated pneumonia was usually mild, only few patients required ventilatory support, 30-day mortality was low (4.2%) and not different to that of patients with pneumonia caused by bacterial (6.2%) or viral pathogens other than influenza (4%), respectively; and 4) concomitant bacterial pneumonia was observed in 21% of patients with influenza and there was a trend for higher mortality in patients with co-infection.
The true incidence and mortality of seasonal influenza-associated CAP is difficult to assess since there is a year-to-year variability in activity of influenza and many previous studies relied on serology with its inherent severe selection bias (only patients with paired serology are detected). Accordingly, aetiological studies in general populations have reported varying incidences ranging from around 6% to 19% depending on the study duration and the methodology used 13–15. Only very few data are available for severe CAP requiring ICU admission. In 16 studies, viral CAP was reported in 1–5%, with influenza being the most common, but it is impossible to retrieve valid data of mortality from these reports 16. In a study addressing elderly patients with severe CAP, the incidence of influenza was found to be trivial, however, the bias of serology-based detection has to be taken into account 17. As far as reported, mortality was always very low.
In a recent study primarily focused on comparing RSV infections with influenza infections, healthy elderly patients (aged ≥65 yrs), high-risk adults (those with chronic heart and lung disease) and hospitalised patients with acute cardiopulmonary illnesses were included using cultures and PCR of nasopharyngeal swabs as well as serology during four consecutive winters. Influenza was found in 5.1%, 5.9% and 12.2%. None of the 44 healthy and high-risk adults with influenza died, as compared with 10 out of 144 (7%) of the hospitalised group with influenza 18. Bacterial infection was indentified in 10% of hospitalised patients with influenza 19.
To our knowledge, only three recent studies have exclusively addressed viral CAP. Based on paired serology, De Roux et al. 6 found influenza in 11.5% of patients with CAP in a 5-yr monocentric study. Due to the methodology applied, mortality could not be assessed. Johnstone et al. 7 investigated viral CAP during a 3-yr period in five hospitals. Influenza diagnosis was based on nucleic acid amplification tests and direct fluorescent antigen assay testing of nasopharyngeal swabs. 19% had viral aetiology, including 4% with a mixed aetiology. Influenza was found in 3.6% of cases. Mortality rate of viral CAP was low (3%). Finally, Jennings et al. 8 investigated viral CAP in a 1-yr monocentric study based on the detection of respiratory viruses in nasopharyngeal swabs by immunofluorescence, culture and PCR. They found influenza in 9.5% of cases, with a mortality of 7%.
Since the classical descriptions of influenza pneumonia from the 1957 and 1968 epidemics 20–22, we are aware only of one single study assessing specifically seasonal influenza pneumonia 23. In this study comprising 35 patients who tested positive for influenza by direct enzyme immunoassay during a 5-yr period, 17 patients had pneumonia. Of these, 10 (58.8%) had to be admitted at the ICU and mortality was high (n = 5, 29%), despite anti-viral treatment in 15 out of 17 patients. Bacterial co-infection was identified in five patients and S. aureus was present in all of them 23. However, the very low number of cases per year makes selection bias quite probable.
A recent review stated that fatal cases of influenza-associated viral pneumonia considered to be primary continue to be identified, but that their incidence appears to be low even in pandemic peaks 24. In line with this notion, we found an incidence of 3.2% and 12% related to patients with a defined aetiology. Of note, the rates varied significantly from year-to-year and had a high seasonal variability. Mortality was 5%, similar to that of two most recent reports 7, 8 and not significantly different from that of the general population of CAP and patients with bacterial CAP.
It has been argued that the majority of influenza deaths are related to secondary bacterial pneumonia 24. It is quite difficult to assess the true incidence of dual bacterial and influenza aetiology in patients with CAP, since both are missed by current investigational methodology in a considerable amount of cases. In our series, such dual aetiology was present in 21% of influenza cases, and this may be an underestimate. As a matter of fact, we found only a nonsignificant slightly higher mortality rate of patients with bacterial co-pathogens.
In our study, S. pneumoniae and not S. aureus was found to be the most frequent bacterial co-pathogen, followed by H. influenzae. This is in accordance with a recent study specifically addressing mixed aetiologies in patients with CAP 25. Variations in primarily involved bacterial pathogens according to antigenic subtypes may be explained by differences in pathogenicity factors 26, 27. In any case, initial empirical antimicrobial treatment has to be administered to all patients with influenza-associated CAP, regardless of concurrent or subsequent detection of bacterial pathogens. An initial anti-microbial treatment covering primarily S. pneumoniae as well as H. influenzae and S. aureus is mandatory. An adequate bacterial coverage seems more important than anti-viral treatment with neuraminidase inhibitors. In fact, there is currently no evidence to recommend anti-viral treatment other than theoretical inference derived from studies of patients with lower respiratory tract infections 28.
Taken together, seasonal influenza-associated pneumonia does not seem to be a particularly deadly condition. Much of the observed excess mortality might be associated with influenza-associated cardiovascular deaths, a notion supported by a reduction in cardiovascular mortality in vaccinated patients 29, 30.
We are not aware of another aetiological study of CAP including such a high number of patients where influenza was systematically investigated using PCR. PCR is more sensitive than virus culture or detection of influenza viruses by immunofluorescence or ELISA. The sensitivity of the PCR assays applied in this study is about 0.1 50% tissue culture infection dose corresponding to 10 genome copies per assay 11. Validation of the assays did not indicate any problems concerning analytical sensitivity, cross-reactivity or detection capability. Therefore, this study represents a new reference for the estimate of the incidence and mortality of seasonal influenza-associated CAP. However, there are several limitations of our study. As in all studies dealing with the aetiology of CAP the availability of respiratory samples is a problem. If only considering patients with a respiratory sample in addition to the throat washings, 2,076 patients (43% of the study population) would have been included in our analysis. We found that excluding these patients made no significant difference for the individual statements, but might weaken the generalisation and conclusiveness of the study and thus included all patients for whom a PCR for influenza had been performed. Another problem is the low number of patients studied with severe CAP requiring ICU admission. However, available series studying patients with severe CAP requiring ICU admission do not suggest influenza to be an aetiology causing excess mortality 16. Another limitation is that we have no data on anti-viral treatment of this population; however, since the German guidelines for management of adult CAP do not recommend such treatment, and since the results of viral investigations were not available during the treatment period, it is highly probable that none of the patients received it.
In conclusion, our data do not support excess mortality in influenza-associated pneumonia or in cases of secondary bacterial infection. Mortality of influenza-associated pneumonia may be limited to the excess incidence of influenza-associated pneumonia cases and deaths during autumn–winter seasons. Bacterial co-pathogens that should always be covered primarily include S. pneumoniae, S. aureus and H. influenzae. Further studies should assess the role of influenza, particularly in patients with severe CAP.
Acknowledgments
CAPNETZ is a multidisciplinary approach to better understand and treat patients with community-acquired pneumonia. The network has only been made possible by the contribution of many investigators. We are especially indebted to the work of the investigators in the local clinical centres who established and kept contact to all practitioners, physicians and respiratory specialists cooperating within the network.
In addition, we would like to acknowledge the work of the central computing unit and the central service unit with A. Sawazki (Institute for Medical Microbiology and Hygiene, Ulm University Hospital, Ulm, Germany) providing excellent technical support.
It is also our responsibility and pleasure to express our appreciation to all clinical physicians and physicians in private practice who saw and identified patients with community-acquired pneumonia for their work dedicated to CAPNETZ.
The collaborating members of the CAPNETZ study group (except the authors) are as follows: G. Rohde, B. Hauptmeier (Dept of Pneumology, Allergology and Sleep Medicine, University Hospital Bergmannsheil, Bochum), T. Schaberg, I. Hering (Dept of Pneumology, Diakoniekrankenhaus, Rotenburg), K. Dalhoff, P. Heyer (Dept of Internal Medicine III, Pulmology, University Hospital Schleswig-Holstein, Lübeck), C. Schumann (Dept of Internal Medicine II, University Hospital Ulm, Ulm), T. Bauer, F. Kunitz (HELIOS Klinikum Emil von Behring, Berlin), H. Schütte, A. Tessmer (Dept of Infectious Disease and Respiratory Medicine, Charité-University Medicine, Berlin), J. Rademacher (Dept of Respiratory Medicine, Hanover Medical School, Hanover), A. Gillissen (Robert-Koch-Klinik, Thoraxzentrum des Klinikums St. Georg, Leipzig), B. Drewelow, J. Majcher-Peszynska (Center of Pharmacology and Toxicology, Institute of Clinical Pharmacology, University of Rostock, Rostock), S. Krüger (Medical Clinic I, University Hospital RWTH Aachen, Aachen), R. Bals (University Hospital Marburg, Marburg), P. Martus (Institute for Biostatistics and Clinical Epidemiology, Charité University Medicine, Berlin), T. Illmann, M. Wallner (2mt Software GmbH, Ulm), G. Barten, L. Gosman (Main Office, Hanover, Germany) and all study nurses.
Footnotes
Support Statement
This network is supported by the German Ministry of Education and Research (Bundesministerium für Bildung und Forschung) Berlin, Germany.
Statement of Interest
None declared.
- Received March 9, 2010.
- Accepted August 17, 2010.
- ©ERS 2011
REFERENCES
