07 February 2013
Low vaccine effectiveness against influenza A(H3N2) virus among elderly people in Denmark in 2012/13 – a rapid epidemiological and virological assessment
In Denmark, the 2012/13 influenza season has been dominated by influenza A(H3N2). We estimated the vaccine effectiveness (VE) of the trivalent influenza vaccine by linking national registers in a test-negative case–control study of patients tested for influenza aged ≥65 years. The adjusted VE against laboratory-confirmed influenza A and B was -11% (95% CI: -41 to 14) and 69% (95% CI: 26 to 87), respectively. Genetic characterisation of the influenza A(H3N2) viruses indicated genetic drift, with seven substitutions at key antigenic sites.
In Denmark, consultation rates for influenza-like illness and the number of patients testing positive for influenza increased in week 51 (starting 17 December) and peaked in week 52 (starting 24 December) 2012. Compared with the previous three influenza seasons, the activity was particularly high among people aged ≥65 years. From week 51, excess mortality was observed in this age group . Influenza A(H3N2) virus has to date been the dominant subtype.
Trivalent influenza vaccine (TIV) was offered free of charge to Danish citizens aged >65 years between weeks 40 and 52. We took advantage of unique national registries to obtain a rapid within-season estimate of influenza vaccine effectiveness (VE) in people of this age group. Furthermore, we describe the vaccination coverage among influenza A patients in intensive care units (ICUs), and characterised circulating influenza A(H3N2) viruses genetically.
Estimating vaccine effectiveness against laboratory-confirmed influenza
Information on patients aged ≥65 years tested for influenza A and B virus by PCR was obtained from the Danish Microbiology Database; information on administered TIV from weeks 39 to 48 was obtained from the Danish vaccination register [2,3]. The study period ran from week 40 (1 October 2012) to week 4 (27 January 2013).
The Danish Microbiology Database comprises real-time data on microbiological diagnostic test results for the entire Danish population. The Danish healthcare sector is public and all influenza testing is done according to national guidelines.
The VE against influenza A and B was estimated in a test-negative case–control design, using the formula (1 − odds ratio) x 100%.
Cases were defined as patients who tested positive for influenza A or B virus. Controls were patients who tested negative for both influenza A and B viruses. As our study is based on results of diagnostic samples, we do not have information on symptoms or indications for testing.
Influenza cases were included when they first tested positive. Patients were considered vaccinated if they received the TIV at least two weeks before the sample was taken; otherwise they were unvaccinated. In sensitivity analyses, the cut-off was changed to three weeks.
Data were analysed in SAS using PROC LOGISTIC. Adjustment was made for age group (65–69, 70–74, 75–80, ≥80 years) and place of sampling (general practitioner vs hospital).
Estimating vaccination coverage
Vaccination coverage among influenza A patients aged ≥65 years in all Danish ICUs was estimated by linking data from the national influenza ICU surveillance system  with that in the vaccination register.
Nucleic acid was extracted from 200 µl of clinical samples (sentinel, surveillance and diagnostic samples), full-length haemagglutinin (HA) genes were sequenced and analysed as described previously . All samples were initially screened for influenza A in a real-time reverse transcription (RT)-PCR reaction targeting the matrix gene . If up to five samples were positive for influenza A(H3) within one week, all samples were set up for sequencing. If more than 5 samples were positive within one week, one sample from each of the five regions of Denmark with an influenza A real-time RT-PCR cycle threshold <35 was picked blindly for sequencing, if available.
Vaccine effectiveness results
A total of 1,443 patients aged ≥65 years were tested during the study period for influenza: 364 and 35 tested positive for influenza A and B, respectively (Table 1). Those who tested negative were considered as controls. Some 95% (1,374/1,443) of the patients were sampled at hospitals. Vaccination coverage increased with age among influenza A cases and controls, and the proportion of patients who tested positive for influenza A also generally increased with age (Table 1).
Table 1. Laboratory-confirmed influenza A and B cases (n=399) and influenza A and B test-negative controls (n=1,044) aged ≥65 years by trivalent influenza vaccination statusa, age group and sex, and vaccination coverage among influenza cases and controls by age group and sex, Denmark, week 40 (1 October 2012)–week 4 (27 January 2013)
Vaccination coverage among the cases was 45%, which was similar to the coverage among the controls (41%), as well as the estimated national coverage among people aged ≥65 years (44%, data not shown). Most of the vaccinated study participants received TIV between weeks 40 to 44; cases of influenza A mainly occurred from week 52 onwards (Figure 1).
Figure 1. Trivalent influenza vaccines received in week 39 (24 September 2012)–week 47 (25 November 2012) and laboratory-confirmed influenza A cases in week 40 (1 October 2012)–week 4 (27 January 2013) among 1,443 patients aged ≥65 years tested for influenza, Denmark
In the study population, VE against laboratory-confirmed influenza A was −11% (95% CI: −41 to 14), whereas VE against laboratory-confirmed influenza B was 69% (95% CI: 26 to 87) (Table 2).
Table 2. Crude and adjusted odds ratios and adjusted vaccine effectiveness estimates against laboratory-confirmed influenza A and B among 1,443 patients aged ≥65 years and tested for influenza, Denmark, week 40 (1 October 2012)–week 4 (27 January 2013)
In the main analysis, patients were considered vaccinated if they received the TIV at least two weeks before the sample was taken. In sensitivity analyses, the cut-off was changed to three weeks, which did not change the estimates.
Vaccination coverage among intensive care unit patients
A total of 53 influenza A patients in ICUs were reported during the study period, of whom at least 22 were vaccinated with TIV at least two weeks before admission. Of the 53 ICU patients, 33 were aged ≥65 years. Samples from 16 of these elderly patients were subtyped: all contained influenza A(H3N2) virus. Of 32 elderly patients with known vaccination status, 15 were vaccinated at least two weeks before admission (47%). Nine were aged 65–69 years, seven were 70–74 years, six were 75–79 years and 10 were ≥80 years. Information on comorbidity was available for 22 of the elderly patients: the two most common comorbidities were cardiovascular and chronic pulmonary disease, reported for 17 of the 22 patients.
Virus characterisation results
The National Influenza Centre in Denmark, Statens Serum Institut, subtyped 487 clinical specimens (those positive for influenza A or B) from week 40 (1 October 2012) to week 4 (27 January 2013). Of these, 395 (81%) were A(H3N2) viruses, 59 (12%) B-Yamagata viruses, 30 (6%) A(H1N1)pdm09 and 3 (<1%) B-Victoria. Based on HA amino acid sequence analysis, the Danish A(H3N2) viruses formed two main clades, A and B (Figure 2). Clade B is within the defined 3C A(H3) phylogenetic clade, where the 2012/13 A(H3N2) vaccine component (A/Victoria/361/2011) is assigned, but the Danish clade A should probably be assigned a new A(H3) clade. Five of the 22 Danish HA sequences included in the phylogenetic analysis, collected from week 46 (11 December 2012) to week 1 (2 January 2013), were from patients who had been vaccinated before week 47: all five were located in clade A (Figure 2). Altogether, seven amino acid substitutions in the HA defined the Danish clade A, compared with egg-grown/tissue grown A/Victoria/361/2011 – at antigenic site B: T128A, R/Q156H and V186G; site A: R142G and N145S; site C: N278K; and site D: S/Y219S. Additional substitutions were Q33R, E/D190D and V347M. The clade B viruses did not possess the substitutions at positions 128, 142 and 347. The clade A viruses have lost the 126 N-linked glycosylation seen in A/Victoria/361/2011 and clade B (predicted by NetNGlyc 1.0 server . For comparison, we included reference HA sequences from influenza A(H3) viruses, obtained from the EpiFlu database of the Global Initiative on Sharing Avian Influenza Data (GISAID) (Table 3).
Figure 2. Phylogenetic tree of 22 influenza A(H3N2) virus sequences coding for 523 amino acids of the viral haemagglutinin, Denmark, week 46 (14 November 2012)–week 2 (13 January 2013)
Table 3. Haemaglutinin sequences of influenza A(H3) virus isolates included in the phylogenetic analysisa
We took advantage of unique newly established national registries to obtain a rapid within-season VE estimate. In the current season, which to date has been dominated in Denmark by influenza A(H3N2) virus, we calculated an adjusted VE point estimate of −11% (95% CI: −41 to 14) against laboratory-confirmed influenza A in patients aged ≥65 years tested for influenza. Given the confidence interval, we cannot exclude a VE of up to 14%, which is still considerably lower than expected. By contrast, vaccination did protect against influenza B (adjusted VE point estimate of 69%). As the National Influenza Centre has subtyped 93% of the influenza A viruses in Denmark as influenza A (H3N2), we expect the poor VE against influenza A to be associated with this subtype. The poor VE against influenza A was supported by a comparable vaccination coverage among ICU patients with influenza A and among Danish people in general in the same age group (≥65 years). We also identified genetic drift of circulating influenza A(H3N2) viruses, which may explain the low VE in elderly people (aged ≥65 years).
The study was observational, with the biases this study type may entail. We were unable to adjust for comorbidity, but we adjusted VE for age group – which was associated with both vaccination uptake and testing positive for influenza A – and place of sampling, which are both likely to be correlated with comorbidity. Although other test-negative VE studies have shown that adjustment for comorbidity only resulted in minor changes in the VE estimates , we cannot exclude the possibility that some residual confounding with comorbidity does exist; however, it is unlikely that the effect of confounding is of a magnitude that would dramatically change the estimates. Furthermore, the VE against influenza B, estimated on only a few cases, was comparable to early estimates from the United States Centers for Disease Control and Prevention . It is possible that some cases and controls classified as unvaccinated had in fact been vaccinated after week 47. However, by way of comparison, in 2011, 95% of the vaccinations were given before week 48.
Our study design has a number of advantages. It was cost-effective because data were obtained from existing sources, and information on vaccination was registered prospectively and independently of outcome. The specificity of the outcome was high and independent of patient recall. The patients tested for influenza may differ from the general population of elderly people but the test-negative design reduces the risk of selection bias, since cases and controls presumably share the same health-seeking behaviours. As 95% of the samples were taken in a hospital setting, the study may indicate a poor protection against severe outcomes of influenza (requiring hospitalisation).
Elderly people who are tested for influenza may represent a more vulnerable group, who have a weaker response to vaccination compared with the elderly population in general; however, if this would explain the findings, we would also expect a low VE against influenza B.
Genetic characterisation revealed that the Danish clade A viruses may be the cause of the low VE observed in elderly people. Four or more substitutions in two or more antibody binding sites are predicted to give an antigenically different virus  – thus the seven differences described for the Danish isolates are a cause for concern. Key substitutions causing the genetic drift are at positions 128 and 142, which are rare substitutions. The A128 substitution causes loss of an N-linked glycosylation site and amino acid changes in the 140–146 region of HA antigenic site A is characteristic for antigenically distinct viruses of epidemic significance . These two substitutions should therefore be considered carefully when antigenic drift is investigated further.
In conclusion, our epidemiological data suggest a low VE against influenza A(H3N2) among elderly people in Denmark; this is in contrast with early VE estimates in the range of 45–55% reported from other studies including all age groups [8,9,12].
Our molecular investigations indicate genetic drift; however, antigenic data on the clade A viruses are needed to document antigenic drift. Therefore, virus neutralisation and haemagglutination inhibition assays are in progress, along with an investigation of the role of the neuraminidase, as the neuraminidase also plays an important role in the genetic and antigenic drift of influenza viruses .
Matching of the influenza A(H3N2) vaccine virus with circulating influenza strains needs further investigation but should be taken into consideration before the coming selection of influenza vaccine strains for the next season, 2013/14. Influenza vaccination is still considered useful among Danish citizens this season due to the high protection against influenza B.
Test results for influenza were obtained from the Danish Microbiology Database (MiBa, http://miba.ssi.dk), which contains all electronic reports from departments of clinical microbiology in Denmark since 2010, and we acknowledge the collaboration with the MiBa Board of Representatives. We also thank the Danish departments of clinical microbiology at Odense, Slagelse, Aalborg, Hillerød and Herlev hospitals for sharing specimens with the National Influenza Centre for the World Health Organization (WHO) in Denmark, Statens Serum Institut.
We greatly value the high technical skills of the technicians Jesper Rønn and Jytte B. Clausen. We acknowledge the authors, originating and submitting laboratories of the sequences from GISAID’s EpiFlu Database (http://www.gisaid.org) (Table 3). We are most grateful for the support of the WHO Collaborating Centre For Reference and Research on Influenza, MRC National Institute for Medical Research, London, United Kingdom.
Conflict of interest
K Bragstad: Conceived the idea for the study and made the genetic and antigenic characterisations, she drafted the first version of the paper, made revisions, and approved the final version of the paper. H-D Emborg: Cleaned the data from the microbiolology database and the vaccination database and made the statistical analysis of VE. She contributed to the interpretation of the data and reviewed the first draft of the paper and approved the final version of the paper. M Voldstedlund: Has established the microbiology database and retrieved the influenza data from the database. She assisted in the data cleaning and interpretation of the results, she reviewed the first draft of the paper and approved the final version of the paper. T K Fischer: Contributed with the interpretation of the virological and epidemiological data and revised the first draft of the paper critically and approved the final version of the paper. S Gubbels: Is in charge of the surveillance of influenza at ICUs in Denmark and contributed with the data on vaccination coverage among ICU patients. She contributed to the interpretation of the results and reviewed and approved the final draft of the paper. B Andersen: Contributed considerably in the laboratory, she approved the final version of the paper. K Mølbak: Has established the microbiology database, he conceived the idea and the design of the VE study and contributed to the interpretation of the results. He revised the first draft of the paper critically and approved the final version of the paper. T G Krause: Conceived the idea and the design of the VE study, she was involved in the data analysis and the interpretation of the results. She wrote the first draft of the paper together with K Bragstad, revised the paper and made the final draft of the paper.
- European Centre for Disease Prevention and Control (ECDC). Weekly influenza surveillance overview. 1 February 2013. Main surveillance developments in week 4/2013 (21–27 January 2013). Stockholm: ECDC; 2013. Available from: http://ecdc.europa.eu/en/publications/Publications/130201_SUR_Weekly_Influenza_Surveillance_Overview.pdf
- Grove Krause T, Jakobsen S, Haarh M, Mølbak K. The Danish vaccination register. Euro Surveill. 2012;17(17):pii=20155. Available from: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20155
- Emborg H-D, Krause TG, Hviid A, Simonsen J, Mølbak K. Effectiveness of vaccine against pandemic influenza A/H1N1 among people with underlying chronic diseases: cohort study, Denmark, 2009-10. BMJ. 2012;344:d7901. http://dx.doi.org/10.1136/bmj.d7901
- Gubbels S, Krause TG, Bragstad K, Perner A, Mølbak K, Glismann S. Burden and characteristics of influenza A and B in Danish intensive care units during the 2009/10 and 2010/11 influenza seasons. Epidemiol Infect. 2012;1–9 [Epub ahead of print]. http://dx.doi.org/10.1017/S0950268812001471
- Bragstad K, Nielsen LP, Fomsgaard A. The evolution of human influenza A viruses from 1999 to 2006: a complete genome study. Virol J. 2008;5:40. http://dx.doi.org/10.1186/1743-422X-5-40 PMid:18325125 PMCid:2311284
- Bragstad K, Martel CJ, Thomsen JS, Jensen KL, Nielsen LP, Aasted B, et al. Pandemic influenza 1918 H1N1 and 1968 H3N2 DNA vaccines induce cross-reactive immunity in ferrets against infection with viruses drifted for decades. Influenza Other Respi Viruses. 2011;5(1):13-23. http://dx.doi.org/10.1111/j.1750-2659.2010.00177.x PMid:21138536
- NetNGlyc 1.0 Server. Center for Biological Sequence Analysis, Technical University of Denmark (DTU), University of Copenhagen. [Accessed 27 Jan 2013]. Available from: http://www.cbs.dtu.dk/services/NetNGlyc/
- Skowronski DM, Janjua NZ, De Serres G, Dickinson JA, Winter A, Mahmud SM, et al. Interim estimates of influenza vaccine effectiveness in 2012/13 from Canada's sentinel surveillance network, January 2013. Euro Surveill. 2013(18(5):pii=20394. Available from: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20394
- Centers for Disease Control and Prevention (CDC). Early estimates of seasonal influenza vaccine effectiveness - United States, January 2013. MMWR Morb Mortal Wkly Rep. 2013;62:32-5. Available from: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6202a4.htm PMid:23325354
- Wilson IA, Cox NJ. Structural basis of immune recognition of influenza virus hemagglutinin. Annu Rev Immunol. 1990;8:737-71.
- Wiley DC, Wilson IA, Skehel JJ. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature. 1981;289(5796):373-8. http://dx.doi.org/10.1038/289373a0 PMid:6162101
- McMenamin J, Andrews N, Robertson C, Fleming DM, Durnall H, von Wissmann B, et al. Effectiveness of seasonal 2012/13 vaccine in preventing laboratory-confirmed influenza infection in primary care in the United Kingdom: mid-season analysis 2012/13. Euro Surveill. 2013;18(5):pii=20393. Available from: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20393
- Gulati U, Hwang CC, Venkatramani L, Gulati S, Stray SJ, Lee JT, et al. Antibody epitopes on the neuraminidase of a recent H3N2 influenza virus (A/Memphis/31/98). J Virol. 2002;76(23):12274-80. http://dx.doi.org/10.1128/JVI.76.23.12274-12280.2002 PMid:12414967 PMCid:136895
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28(10):2731-9. http://dx.doi.org/10.1093/molbev/msr121 PMid:21546353 PMCid:3203626