Evidence and policy for influenza control

H Kelly1,2, B J Cowling (bcowling@hku.hk)3 1. Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia 2. National Centre for Epidemiology and Population Health, Australian National University, Canberra, Australia 3. Division of Epidemiology and Biostatistics, School of Public Health, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong Special Administrative Region, China

This edition of the journal includes two studies related to the control of influenza, one on neuraminidase inhibitor (NI) resistance [1] and the other on the effectiveness of trivalent influenza vaccine in the United Kingdom in 2012/13 [2].Neuraminidase inhibitors (NIs) are the mainstay in influenza treatment and vaccination is the mainstay of prevention.It is thus important to monitor the effectiveness of both interventions over time.The current NI study demonstrates that mutations which may have had clinical significance for previously circulating seasonal influenza A(H1N1) viruses may not be clinically significant for influenza A(H1N1) pdm09 viruses, highlighting the importance of continued monitoring of NI resistance.Of equal importance is the continued monitoring of NI effectiveness [3].
Influenza is a common disease with the annual risk of influenza virus infection exceeding 20% in some years [4,5].However the great majority of influenza virus infections do not present as the classical triad of fever, cough and fatigue [6][7][8], and a substantial proportion of infections, perhaps even more than half, are asymptomatic [4,5].Even symptomatic illnesses are generally self-limiting.However a small proportion of persons with influenza virus infections will require admission to hospital, intensive care and a smaller proportion will die [9].These outcomes are uncommon and are influenced by age, with increased risk at the two extremes of life, and the presence of co-morbidities [10].For instance, unadjusted annual risk estimates of laboratory-confirmed influenza hospitalisation in hospitals from the Emerging Infections Program in the United States between 2005 and 2011 ranged from 20 to 72 per 100,000 for children up to the age of four years, from 16 to 76 per 100,000 for adults aged at least 65 years, but only from 5 to 14 per 100,000 for adults aged 20 to 64 years, although higher in the first year after influenza A(H1N1)pdm09 emerged [11].About 10-30% of people hospitalised with influenza will require intensive care [12][13][14], and about 3-10% of patients hospitalised with laboratory confirmed influenza will die [13][14][15].
Because serious outcomes are relatively rare, randomised controlled trials (RCTs) in ambulatory settings for the treatment of influenza with NIs or the prevention of influenza by vaccination have not been designed with sufficient power to examine these outcomes.RCTs of antiviral drugs [16] and vaccines [17] and have shown efficacy against suspected and laboratory-confirmed influenza acquired and managed in the community but there are no RCTs investigating outcomes of hospitalisation or death due to laboratory-confirmed influenza.
It is generally acknowledged that when outcomes are rare, the RCT is not necessarily the study design of choice.The classic case-control study, in which cases and controls are ascertained retrospectively, has often been the preferred alternative design.A variation of the classic design has become increasingly popular for studying vaccine effectiveness (VE) against specific outcomes.In what is referred to as the case-testnegative design, patients with respiratory symptoms are ascertained prospectively, and vaccine coverage is compared between those who test positive and those who test negative for influenza, adjusting for potential confounders [18].The second study of influenza in this issue of the Eurosurveillance uses the case-test-negative design in pooled community-based studies from the United Kingdom to estimate influenza VE against medically-attended respiratory disease confirmed as influenza.It reports point estimates of 73%, 26% and 51% against influenza A(H1N1), A(H3N2) and B, respectively [2].These results confirm a number of other findings of low VE against influenza A(H3N2) in recent years [19][20][21], attributed to mismatch between the vaccine and circuiting strains [19].They also highlight the importance of monitoring not only the antigenic match, as determined by serological assays, but also the genetic relatedness of circulating and vaccine viruses.
The case-test-negative design is also being increasingly used for studies of hospitalised patients, using PCR-confirmed influenza as an outcome.These studies suggest that inactivated influenza vaccines decrease the risk of hospital admission for laboratory-confirmed influenza by about half [22,23], although lower estimates have been reported for the protection against influenza A(H3N2) in the elderly [24] and higher estimates for protection against influenza A(H1N1)pdm09 [25].A 50% decrease in risk is similar to effectiveness estimates from community observational studies using the same design [26,27], and efficacy estimates from meta-analyses of community-based trials [17].
For information on the effectiveness of NIs among hospitalised patients, we likewise need to rely on observational studies.A recent review critically examined published cohort studies assessing oseltamivir treatment for laboratory-confirmed influenza and found evidence suggesting protection against mortality in four studies, all of which were judged by the review to be of reasonable quality, and between which there was no statistical heterogeneity [28].
Even the best designed observational studies may be subject to residual bias, suggesting the need for RCTs.However RCTs of NIs in outpatients with increased risk of complications, and in patients hospitalised soon after onset of symptoms may no longer be feasible because oseltamivir is the accepted front-line treatment in groups of patients with suspected or confirmed influenza [29][30][31] and such trials may no longer be granted ethical approval.The same argument applies to influenza vaccination for people aged 65 years and over.For these reasons, better quality data are unlikely to be derived from RCTs, so that observational studies might do well to follow published quality guidelines in an effort to improve VE estimates [32].Doubt has been cast on the efficacy of influenza vaccines against serious outcomes in the elderly because of the absence of trial data [33].Similar discussions are occurring about the efficacy of anti-viral medication [3,16].At the same time, it is being increasingly recognised that influenza infection in the community is common and that infections are associated with a wide clinical spectrum, but the serious consequences of infection are generally uncommon, and often rare, in healthy young people [5].Improved policies for the control of influenza virus infection should acknowledge the wide clinical spectrum resulting from infection, so that prevention or treatment of serious outcomes will be attempted when serious outcomes are more likely.Such policies should use data from observational studies where trial data are absent.

Research articles
Effectiveness of trivalent seasonal influenza vaccine in preventing laboratory-confirmed influenza in primary care in the United Kingdom: 2012/13 end of season results

Introduction
The 2012/13 influenza season in the United Kingdom (UK) was unusually long with elevated levels of activity persisting from week 50 to 16.In England, Northern Ireland and Wales, the season was dominated initially by circulation of influenza B virus, with school outbreaks in the period before Christmas.This was followed by influenza A(H3N2) virus circulation particularly in the New Year and spring with influenza outbreaks in often highly vaccinated care home populations [1].Scotland presented a different picture with influenza activity initially dominated by influenza A(H3N2) followed by influenza B virus circulation.
The occurrence of late season influenza A outbreaks in much of the UK some months after the completion of the 2012/13 influenza vaccine campaign led to questions being raised about waning intra-seasonal vaccine protection.A similar observation of late season influenza A(H3N2) outbreaks in care home settings in 2011/12 was accompanied by the observation of significant intra-seasonal waning in protection for those vaccinated more than three months previously [2].
Mid-season estimates from the UK and elsewhere were published in January 2013 with vaccine effectiveness (VE) against all influenza types ranging from 45% in Canada to 50% in a European study and 51% in the UK [3][4][5].This study presents the end-of-season VE for the 2012/13 seasonal TIV in preventing medically attended confirmed influenza A(H3N2), A(H1N1)pdm09 and B virus infection using the established primary care sentinel swabbing surveillance schemes in the UK [2,3,6].It also examines the protective effect of vaccination measured at different points during the season and by time since vaccination, to determine if there is any evidence of intra-seasonal waning protection.[7].

Study population and period
The study period ran from 1 October 2012 to 24 April 2013.Patients were swabbed as part of clinical care, with verbal consent.Cases were defined, as persons presenting during the study period in a participating general practitioner (GP) practice with an acute influenza-like illness (ILI) who were swabbed and then tested positive for influenza A or B. ILI was defined as an individual presenting in primary care with an acute respiratory illness with physician-diagnosed fever or complaint of feverishness.Controls were individuals presenting with ILI in the same period who were swabbed and tested negative for influenza.
A standardised questionnaire was completed by the GP responsible for the patient during the consultation.Demographic, clinical and epidemiological information was collected from cases and controls, including date of birth, sex, defined underlying clinical risk group, date of onset of respiratory illness, date of specimen collection, and influenza vaccination status for the 2012/13 season with vaccination dates.

Laboratory methods
Laboratory confirmation was undertaken using realtime polymerase chain reaction (RT-PCR) assays capable of detecting circulating influenza A viruses, influenza B viruses and other respiratory viruses [8,9].Samples in England were sent to the PHE Microbiology Services, Colindale (RCGP scheme) or one of the specialist PHE microbiology laboratories (SMN scheme).Samples in Northern Ireland were sent to the Regional Virus Laboratory, Belfast, in Scotland to the West of Scotland Specialist Virology Centre, Glasgow (HPS scheme), and in Wales to the Public Health Wales Specialist Virology Centre, Cardiff.
Further strain characterisation was also performed.Influenza viruses were isolated in Madin-Darby canine kidney (MDCK) or MDCK-SIAT1 cells from all RT-PCR positive samples from England as previously described [10].Virus isolates with a haemagglutination titre ≥40 were then characterised antigenically using post-infection ferret antisera in haemagglutination inhibition (HI) assays, with guinea pig (A(H3N2) viruses) or turkey (A(H1N1)pdm09 and influenza B viruses) red blood cells [11].Nucleotide sequencing of the HA1 region of the haemagglutinin (HA) gene of a subset of influenza B viruses was performed (primer sequences available on request), and phylogenetic trees were constructed with a neighbour-joining algorithm available in the  1).

Statistical methods
Persons were defined as vaccinated if the date of vaccination with the 2012/13 TIV was 14 or more days before onset of illness.Those in whom the period between vaccination and onset of illness was less than 14 days were excluded from analysis.If the date of vaccination was missing, as the 2012/13 campaign occurred before influenza circulation was common, it was assumed that TIV vaccination was more than 14 days before onset date.Those with a missing date of onset or an onset date more than seven days before the swab was taken were excluded.
VE was estimated by the test-negative case-control (TNCC) design [12].In this design VE is calculated as 1 -(odds ratio) obtained using multivariable logistic regression models with influenza A or influenza B PCR results as outcomes and seasonal vaccination status as the linear predictor.Influenza A results were also further categorised for A(H3N2) and A(H1N1)pdm09 viruses.In the analyses evaluating VE for a specific type or strain, those positive for other types were excluded.Age (coded into five standard age groups, <5, 5-

Strain characterisation
Antigenic analysis by HI assay of influenza A(H3N2) viruses isolated from positive samples submitted through the RCGP scheme, demonstrated that these viruses were antigenically homogeneous, and closely related antigenically (<fourfold differences in HI assays for all 89 tested) to the cell-propagated H3N2 vaccine strain, A/Victoria/361/2011 [1].The relatively fewer influenza A(H1N1)pdm09 viruses isolated in 2012/13 were closely related antigenically to the A(H1N1) pdm09 2012/13 vaccine strain, A/California/7/2009, though six of thirty isolates did show reduced reactivity in antigenic characterisation assays with antiserum raised against influenza A/California/7/2009 (>fourfold difference in HI assays).

Model fitting for vaccine effectiveness estimation
When estimating vaccine effects, age group, sex, time period (defined by month of sample collection) and surveillance scheme were adjusted for in a multivariable logistic regression model.Although all these variables, except sex, were significantly associated with having a positive swab, only age group was a confounder for the vaccine effects (changed the estimate by more than 5%).Information on risk group was missing for 182 of 3,286 samples (5.5%) and was therefore not included in the final model.If risk group was included, it was found not to be associated with being positive and the VE estimates remained similar.
Tables 3 and 4 show VE estimates against influenza A(H3N2), A(H1N1)pdm09 and B according to vaccination status and time since vaccination and period.
For influenza A(H1N1)pdm09, VE showed evidence of a decline by time since vaccination (Table 4).For influenza A(H3N2), there was also evidence of a decline in VE from 50% (95% CI: 16 to 71) within three months of vaccination to 2% (95% CI: -49 to 36) after three months.This decline was not, however statistically significant (p=0.25) and was less apparent when looking at VE by period (Table 3).Assessing time since vaccination with those with a missing date of vaccination excluded gave similar results with a decline from 100% (95%CI: 56 to 100) to 63% (95% CI: -7 to 87) for influenza A(H1N1) pdm09 and from 41% (95%CI:0 to 66) to 5% (95% CI: -51 to 41) for influenza A(H3N2).
The adjusted age-specific estimate for influenza A protection was lower in the oldest age group (≥65 years compared to other ages) (Table 4), however, the observed differences were not significant.VE in the vaccine target group (aged ≥65 years or in a risk group) was 13% (95% CI:-44 to 67) due to the low VE estimate in those aged ≥65 years.VE also showed some Numbers and row percentages (to indicate positivity ratesa) are shown.For example of those 294 swabbed aged <5 years, 72.8% were negative, 17.0% had influenza B and 10.5% had influenza A.
Differences between cases and controls for all variables in this table were statistically significant, chi-squared test.a Two individuals positive for influenza A(H3N2) and B and one individual positive for A(H1N1)pdm09 and B, are included in both the influenza B and influenza A columns.For the totals in this column, these individuals are only counted once.b 25 influenza A cases were of unknown strain, these are included in influenza A VE analysis but not the strain specific analyses.Also two individuals positive for influenza A(H3N2) and B and one individual positive for A(H1N1)pdm09 and B are included in both the influenza B and influenza A columns, which is why the total adds to 3,289 for controls, influenza A and influenza B. c Where a date of vaccination was missing (n=150) this was estimated by assuming vaccination was on 20 October 2012, the median time of vaccination in controls with onset in 2013.variability across the schemes although this difference was not significant.

Vaccine effectiveness against influenza B infection
The adjusted VE of TIV against influenza B was 51% (95% CI: 34 to 63).VE was 57% within three months of vaccination and non-significantly lower at 42% after three months (Table 4).VE did not vary by age group or scheme.

Discussion
In this study we found moderate effectiveness of 2012/13 TIV against laboratory-confirmed influenza B and good protection against influenza A(H1N1)pdm09 infection.However, VE against influenza A(H3N2) infection was poor at only 26% (95% CI: -4 to 48).We also found a non-significant trend that effectiveness waned by time since vaccination for influenza A(H3N2), which is consistent with the waning seen against influenza A(H3N2) for the 2011/12 TIV vaccine in the previous season where it reduced from 53% (95% CI: 0 to 78) within three months of vaccination to 12% (95% CI: -31 to 41) after three months [2].The point estimate for 2011/12 was also similar for influenza A(H3N2) at 23% (95% CI: -10 to 47), although VE was higher in 2011/12 against influenza B at 92% (95% CI: 38 to 99) compared to 2012/13.The VE against A(H1N1)pdm09 is consistent with that seen with monovalent pandemic vaccine (adjuvanted) in 2009/10 and with TIV in 2010/11 [6,7].
Influenza vaccine strains are propagated in eggs during the vaccine manufacturing process.It has been reported that propagation of the A/Victoria/361/2011(H3N2)-like vaccine viruses for vaccine production resulted in antigenic changes in the virus resulting from adaptation to the growth in eggs, although circulating viruses were closely related antigenically to the cell-propagated influenza A(H3N2) vaccine strain, A/Victoria/361/2011 [13].Following these observations the influenza A(H3N2) vaccine component for use in the 2013/14 season (northern hemisphere winter) has been updated to recommend an influenza A(H3N2) virus antigenically like the cell-propagated A/Victoria/361/2011 prototype strain (such as A/Texas/50/2012) [13].The majority of influenza B virus isolates in 2012/13 were characterised as belonging to the B/Yamagata/16/88-lineage, as does the 2012/13 influenza B vaccine strain, B/ Wisconsin/1/2010.However, genetically, the HA genes of the majority of B-Yamagata strains fell within a genetic clade (clade 2), which in HI assays are antigenically distinguishable from B/Wisconsin/1/2010 (genetic clade 3)-like viruses.This provides an explanation for the lower VE observed against influenza B in 2012/13 compared to 2011/12.Consequently the influenza B vaccine component recommended for use in 2013/14 has also been updated, to a B/Massachusetts/2/2012 (clade2)-like virus [13].
When stratifying VE by age and scheme, VE is estimated with lower precision.There were no significant differences in VE by age or scheme although the point estimate for VE against influenza A was negative for the SMN scheme and also for the over 65 years age group.These differences are likely to be chance fluctuations due to small numbers and emphasise the need for large numbers of swabs for precise estimates for such subgroup analyses.This is now the fourth season in which a pooled UK VE analysis has been performed using the TNCC design with mid-season estimates also produced for 2009/10, 2010/11 and 2012/13 [2,3,6,7,14].Results from the RCGP scheme have also been published for 2005/06, 2006/07 and 2007/08 [15].The results of each season have been consistent with those published from other countries and from pooled European analyses but often with greater precision in the UK due to the large numbers of swabs.The mid-season 2012/13 results gave VE against influenza A of 49% (95% CI: -2 to 75) and influenza B of 52% (95% CI: 23 to 70), which was similar to other early season results from Europe and Canada [4,5], and also similar to the end of season result of 51% (95% CI: 34 to 63) for influenza B, but higher than the end of season result of 35% (95% CI: 11 to 53) for influenza A. The TNCC design is now the most commonly used method for estimating the VE of influenza vaccines.This reflects the advantages of the method in terms of its simplicity and the fact that those that test negative form an excellent control group as they are well matched on propensity to consult a GP.Further discussion of the methodological issues have been published previously [12,16,17] and a recent paper has demonstrated the methods validity compared to placebo controlled clinical trial results [18].
The intra-seasonal waning of VE against influenza A(H3N2) seen in 2011/12 in the UK is supported by the estimates seen this year, albeit non-significant.Carehome outbreaks late in the season both in 2011/12 and 2012/13 also support this observation [1,19], as did similar findings in other countries in 2011/12 [20].With a new influenza A(H3N2) and B strain recommended for 2013/14 and with the introduction of a trivalent live attenuated intranasal vaccine for all children aged 2-3 years and up to 10 years of age in parts of the UK [21] monitoring of VE remains an essential part of influenza surveillance.
collection.Funding: The infrastructure and methodology to enable the work was developed as part of a European Centre for Disease Control and Prevention (ECDC) funded project "I-MOVE".The I-MOVE group continues to be co-ordinated by Epi-Concept (http://www.epiconcept.fr/).

Introduction
Antiviral drugs are essential in the treatment of influenza disease, especially for severe cases, and can play a very important role in the response to the early phases of a pandemic, when a suitable vaccine may not be available and may take several months to develop [1][2][3].Due to rapid emergence and spread of strains highly resistant to adamantanes, these first generation antiviral drugs targeting the influenza virus M2 protein, have been replaced by neuraminidase inhibitors (NAIs), oseltamivir and zanamivir [4].Before their introduction in 1999, analysis of over 1,000 clinical specimens confirmed the general lack of naturally occurring resistance to NAIs [5].Reduced inhibition by NAIs without treatment pressure was rare in influenza viruses in 2002 [6], although reports of resistance to NAIs associated with treatment appeared subsequently [7,8].In 2007/08, oseltamivir-resistant seasonal influenza A(H1N1) virus variants associated with the H275Y neuraminidase (NA) mutation (N1 numbering) and unrelated to drug use, emerged and spread globally.During 2008/09, these resistant variants became the predominant seasonal A(H1N1) viruses worldwide [9].Since late 2009, A(H1N1) seasonal viruses have no longer been in circulation, having been replaced by the swine-origin A(H1N1)pdm09 pandemic virus subtype.The influenza A(H1N1)pdm09 viruses were susceptible to the NAIs oseltamivir and zanamivir, and carried an NA gene segment which belonged to the same genetic lineage as the NA in the Eurasian swine influenza viruses [10].Since the beginning of the A(H1N1) pdm09 pandemic, oseltamivir resistance has remained but with low prevalence.Several reports describe oseltamivir-resistant A(H1N1)pdm09 viruses bearing the H275Y NA substitution, isolated from patients both treated and untreated with NAIs [11][12][13][14][15][16], as well as sporadic clusters of patients who acquired infection with these resistant viruses by human-to-human transmission [13,[17][18][19].These reports raise concerns that the prevalence of viruses exhibiting reduced inhibition by NAIs may increase in the future, and thus, monitoring for the emergence of such viruses is an essential part of national and international surveillance and prevention programmes.
Various amino acid substitutions in the viral NA have been associated with reduced inhibition by NAIs in both seasonal and pandemic A(H1N1) viruses, like the aforementioned H275Y or the E119G/V and I223M/V mutations among others (N1 numbering) [9].The Y to H substitution in the residue number 155 of the viral NA (N1 numbering) was first described by Monto et al. in a seasonal influenza A(H1N1) virus (A/Hokkaido/15/02 strain) isolated during a study describing the global prevalence of resistance to NAIs during the first three years of their use [6].Despite the fact that the Y155H substitution is uncommon and has not been included in the main surveillance programmes of the World Health Organization (WHO) [20], it has been listed as a mutation that should be routinely monitored, as recently published by the Antiviral Susceptibility Task Group of the European Influenza Surveillance Network (EISN), Community Network of Reference Laboratories for Human Influenza in Europe (CNRL), coordinated by the European Centre for Disease Control and Prevention (ECDC) [21].Here we present what is, to the best of our knowledge, the first report of pandemic human influenza A(H1N1) viruses bearing the Y155H amino acid substitution in the NA protein and their susceptibility to NAIs.

Methods
Representative influenza virus isolates and positive respiratory clinical specimens gathered through the Spanish Influenza Surveillance System (SISS) during the 2012/13 influenza season in Spain were sent to our laboratory for complete characterisation.Briefly, the complete haemagglutinin (HA) and NA genes were amplified through RT-PCR and subsequently sequenced following previously described methods [22,23].We then performed a phylogenetic analysis of the HA sequences to allocate each virus in the different genetic clusters [24].The sequences of the NA (N1 numbering) were also analysed for the presence of mutations related with reduced inhibition by NAIs (V116A, I117V, E119G/A/V, Q136K, Y155H, D199G/N, I223M/V/K/R, S247N, K262R, H275Y and N295S) [23].For the sake of clarity, N1 numbering will be used throughout.Selected isolates, including among others those bearing any of these mutations, specimens suspected to have reduced susceptibility to NAIs or from severe cases before start of oseltamivir treatment, were passaged on MDCK cells and their phenotype was analysed using the well described MUNANA assay as recommended in the WHO guidelines [25].Resistant and sensitive virus controls distributed by the International Society for Influenza and Other Respiratory Viruses (ISIRV) were included in all assays.
The NA gene of the A(H1N1)pdm09 viruses containing the Y155H mutation was amplified and subsequently sequenced with the Illumina MiSeq high-throughput sequencing platform.A control virus (A/StPetersburg/100/2011-like) previously characterised in our laboratory as bearing a tyrosine at position 155 was also included.A 1,000 bp NA gene fragment containing the position 155 was amplified through RT-PCR [22,23].DNA libraries were prepared from the amplicons (starting DNA quantity: 1 ng from each) following the Nextera XT (Illumina) standard protocol.The libraries were sequenced in one Illumina MiSeq run of 2x150 (v2) format and represented 7% of the total of samples of the pool which was sequenced.The output was analysed with FastQC and NGStoolkit software for quality assurance.Alignment was performed with BWA alignment software (v.0.6.2) and the variant calling was performed with SAMtools mpileup software (v0.1.18)and a perl script specifically developed for this purpose.
In order to search for other Y155H A(H1N1)pdm09 NA sequences, partial or entire A(H1N1)pdm09 NA sets of nucleotide and amino acid sequences were downloaded from the GISAID EpiFlu (http://platform.gisaid.org) database (11,548 nucleotide and 11,871 amino acid sequences available on 2 December 2013).We gratefully acknowledge the originating and submitting laboratories who contributed sequences to that database.Sequences were aligned using Muscle software (v3.8.31).Sequences with a substitution in the residue Y155 were analysed and quantified with an R script specifically developed for this purpose.

Haemagglutinin and neuraminidase genetic analysis
From the beginning of the 2012/13 influenza season until week 20 (2013), we genetically characterised a total of 227 influenza A virus isolates, which were classified according to the phylogenetic analysis of the HA gene sequence (data not shown).Among them, 175 were A(H1N1)pdm09 viruses (143 A/StPetersburg/27/2011like and 32 A/StPetersburg/100/2011-like).NA gene analysis was performed on 47 A(H1N1)pdm09 viruses, 45 of which did not have any mutations associated with reduced inhibition by NAIs.However, two viruses bearing the Y155H substitution were detected.These two viruses had been isolated from two severely ill hospitalised patients who had been diagnosed with severe respiratory syndrome and pneumonia and required admission to the intensive care unit (ICU) (Figure 1 and Table 1).Influenza A(H1N1)pdm09 infection had been diagnosed in both cases at primary healthcare institutions based on nasopharyngeal aspirates obtained before the start of the oseltamivir treatment.These samples were sent to the regional laboratories for influenza surveillance for viral isolation.The resulting isolates (sample ID 13508 and 13752 from Patient 1 and Patient 2 respectively; Table 1) were then sent to our laboratory for further characterisation.Once we detected the Y155H substitution in both isolated viruses (A/Galicia/508/2013 and A/Extremadura/752/2013 from Patient 1 and Patient 2, respectively; Figure 1), RNA directly extracted from the corresponding nasopharyngeal aspirates was required to confirm the substitution in non-cultured viruses.Original RNA extract from the direct sample specimen was only available from Patient 2 but not Patient 1.The Y155H substitution was confirmed in the NA sequence of the original specimen from Patient 2, presenting clear evidence that this substitution had not been generated during primary cell culture isolation.

MUNANA neuraminidase inhibition assay
In order to obtain a sufficiently high virus titre to perform the NA enzyme inhibition assay [25], virus isolates from Patient 1 and Patient 2 were passaged in MDCK cell cultures.The sequence of the NA gene of the new viral stocks was analysed again before performing the NA enzyme inhibition assays and the presence of Y155H substitutions was confirmed.NA enzyme inhibition assays were subsequently performed.In those, neither of the two Y155H pandemic viruses showed a phenotype of reduced inhibition by NAIs (Table 2).The IC 50 values that were obtained for the Y155H viruses, were in the same range as the ones obtained for the other 21 sensitive viruses that were tested in our laboratory during the 2012/13 season (mean IC 50 : 0.6±0.28,range: 0.17-1.60).In order to confirm these results, viral stocks of both cases were sent to the Respiratory Virus Unit at Public Health England (PHE) in London, United Kingdom.Phenotypic data from both laboratories closely correlated, confirming the absence of reduced inhibition by NAIs in both Y155H A(H1N1)pdm09 viruses (Mean IC 50 of 0.7 for oseltamivir and 0.4 for zanamivir in the case of A/Extremadura/752/2013, and of 0.3 for both NAIs in the case of A/Galicia/508/2013).

High-throughput sequencing of the neuraminidase gene of the Y155H viruses
Original virus isolates from Patient 1 and Patient 2, along with the RNA from the original clinical specimen from Patient 2 and a wild-type control virus (A/ StPetersburg/100/2011-like) were included in the highthroughput sequencing analysis.Around 300,000 high quality (70% of the bases with more than 20 Phred quality) reads per sample were obtained from the highthroughput sequencing run.Samples were sequenced to between 30,708 times and 64,154 times average depth of coverage.The first nucleotide of the triplet coding for NA residue 155 was read 35,488 times for A/Galicia/508/2013.Of those, 35,440 (99.86%) contained the codon CAT, coding for a histidine at amino acid position 155, and only 39 (0.11%) contained the wild-type codon TAT, coding for a tyrosine (Sample 508; Figure 2).In the case of A/Extremadura/752/2013, 20,136 reads were obtained for the first nucleotide of the codon of interest, of which 20,124 (99.94%) contained the codon CAT coding for a histidine and 10 (0.049%) contained the wild-type codon TAT (Sample 752; Figure 2).Similar results were observed in the RNA directly extracted from the clinical sample specimen from Patient 2 (Sample 948; Figure 2).In contrast, the wild-type A/StPetersburg/100/2011-like control virus (Sample C077; Figure 2) contained the codon TAT coding for a tyrosine in 35,281 of 35,338 total reads (99.84%).In this sample, 53 (0.15%) reads with the Y155H substitution were also detected.These results correlated with those obtained in the Sanger sequencing (Figure 1) and confirmed that the Y155H viruses can be considered as pure H155 populations.and from different countries in Africa, Asia, Australia, Europe and North America.Information on their phenotype of susceptibility to NAIs was only available for a five of these samples, which had all been reported as not showing reduced inhibition by NAIs in the GISAID EpiFlu database.We also found 10 sequences with a Y to F substitution at position 155, and one with a Y to C substitution.

Discussion
According to data obtained from the EISN network during the 2012/13 influenza season in Europe (up to week 20 in 2013), 11 of a total of 614 A(H1N1)pdm09 viruses tested for antiviral susceptibility carried the NA H275Y amino acid substitution [26].These 11 viruses were detected mainly in oseltamivir-treated hospitalised patients, some of whom were immunocompromised [26].Excluding the two isolates bearing the Y155H substitution described here, none of the remaining 601 A(H1N1)pdm09 viruses tested for neuraminidase inhibitor susceptibility showed genetic or phenotypic evidence of highly reduced or reduced inhibition [26].
To the best of our knowledge, this is the first report of the presence of the Y155H amino acid substitution in circulating pandemic A(H1N1) viruses.However, we found 56 NA amino acid sequences with the Y155H substitution among the 11,871 A(H1N1)pdm09 NA records available at the GISAID EpiFlu database by the date of our analysis.These sequences had been reported to the database before and during the 2012/13 season from different countries in Africa, Asia, Australia, Europe and North America.This suggests that A(H1N1) pdm09 viruses containing Y155H had already been circulating worldwide for several seasons, albeit at a very low prevalence.
It should be highlighted that the two influenza A(H1N1) pdm09 viruses with the Y155H mutation were detected in samples obtained before the start of treatment with oseltamivir, and they are thus naturally occurring variants not associated with a selective pressure derived from the usage of NAIs.Monto et al. described for the first time the Y155H substitution in a Japanese seasonal influenza A(H1N1) virus strain (A/Hokkaido/15/02) isolated three years after the licensing of oseltamivir and zanamivir [6].This strain exhibited a 123-and a 555fold increase in the mean IC 50 values for zanamivir and oseltamivir, respectively [6].However, the two Y155H viruses described here behaved like the sensitive  control strains and did not show oseltamivir and zanamivir IC 50 values in the highly reduced or reduced inhibition range (Table 2).These results are in accordance with the fact that the two patients fully recovered from disease under oseltamivir treatment.
The results of the high-throughput sequencing analysis of the NA of the Y155H A(H1N1)pdm 09 viruses revealed that they can be considered as pure H155 populations, as only a very low level of wild-type Y155 background was observed (<0.2% of the total reads).These results allow us to discard the possibility that the sensitive phenotype observed in the MUNANA assays for the Y155H viruses could have been conferred by a wild type Y155 background with a higher NA activity, as it has been recently reported for the R292K substitution in H7N9 viruses [27,28].
Therefore, it can be concluded that, in contrast to what was observed for the Japanese seasonal influenza A(H1N1) A/Hokkaido/15/02 strain, a change from Y to H in the residue 155 of the NA does not seem to confer any reduced inhibition by NAIs in A(H1N1)pdm09 viruses.This is somewhat unexpected, considering the significant impact that this substitution has on the A/ Hokkaido/15/02 seasonal A(H1N1) virus, as shown by  McKimm-Breschkin et al. in a recent publication [29].
McKimm-Breschkin et al. show that the Y155H mutation dramatically reduces susceptibility to all NAIs, and that it also reduces plaque size and affects the activity, stability, substrate affinity and pH profile of the NA [29].However, the different behaviour of our A(H1N1) pdm09 Y155H mutants in the inhibition assays could be explained by distinct structural features of their NA.Qing Li et al. show that the A(H1N1)pdm09 NA does not have the 150-cavity characteristic of other group 1 neuraminidases, including seasonal A(H1N1) viruses [30].
Their results, together with other similar work related not only to NA but also to HA structures, show that the structures of both proteins in the 2009 pandemic virus are distinct from the corresponding structures in the seasonal virus [31,32].
In the case of severe influenza disease, a decision to use the current neuraminidase inhibitors must be made urgently.In these cases it is essential to rule out the presence of a substitution in the NA of the infecting virus which could compromise the effectiveness of the treatment.Our data imply that in a patient with severe influenza disease caused by an A(H1N1)pdm09 virus carrying the Y155H substitution in the neuraminidase, oseltamivir and zanamivir can be chosen as the therapeutic tool.In conclusion, we believe that this report can contribute to a better understanding of the biological significance of amino acid substitutions in the influenza virus NA and HA in relation to susceptibility to NAIs.

Introduction
This study is set in the context of a large outbreak in London of Salmonella Enteritidis phage type (PT)14b with antimicrobial resistance to nalidixic acid and reduced susceptibility to ciprofloxacin (SE PT14b NxCpL).On 16 September 2009, three large London hospitals informed the North West London Health Protection Unit (NWLHPU) of a cluster of patients presenting with diarrhoea and vomiting.All cases had consumed products from a takeaway restaurant, up to 55 hours before becoming ill.The first person with enteric illness presented to the hospital on 9 September.Analysis of stool samples from the initial patients identified S. Enteritidis PT14b.Immediate public health measures were taken on the day of reporting and the takeaway shop voluntarily closed on 16 September.
Food poisoning outbreaks related to non-typhoidal Salmonella can result in high attack rates [1] and serious complications with associated excess morbidity and mortality amongst those affected [1][2][3].The investigation of these outbreaks requires microbiological and epidemiological evidence to ensure appropriate control measures and inform public health action [4].
When a cohort cannot be precisely identified, casecontrol studies are the method of choice.The feasibility of this study design is determined by available resources and the ability to recruit suitable controls from the population at risk.Greater social mobility and the use of mobile phones make traditional methods of control selection through random digit phone dialling more difficult and less valid, introducing sizable selection bias [5].Case-nominated controls risk 'overmatching' on the causal exposure, and use of population registers is often not possible, as they do not always identify the population at risk in the outbreak.
Alternative study designs have been used, such as, case-crossover studies [6], case severity studies [1] and serotype case-case studies [5,[7][8][9].In addition, analyses-of-sales studies have been reported [10,11].These were hypotheses-generating consumer record analyses [10], and a comparison of the probability of kebab consumption based on information from the kebab shop owner with exposure information from the cases [11].However, many of these approaches are contextual and cannot be easily adapted for other outbreak situations.
Based on the initial report of this large cluster of cases with gastrointestinal illness with exposure to food from a north west London takeaway restaurant we sought to establish epidemiological and microbiological evidence for the source and vehicle of the outbreak.
In addition, we explored the feasibility and validity of a new analytical approach, using till receipts from the restaurant to both estimate exposures in the population at risk, as well as sales volumes as an exposure in an ecological case-crossover approach.

Epidemiological investigation
The outbreak was described in time, person and place to generate hypotheses about the possible exposures and exposure mechanisms.Cases were defined as below (box).Following the initial notification, a casefinding exercise was conducted.This involved notification requests from local frontline clinicians (e.g.general practitioners, hospital doctors and microbiologists) for cases with an epidemiological link to the takeaway restaurant.Some reported cases also informed on known fellow diners that became ill.Additionally, national reference laboratory reports for all S. Enteritidis PT14b cases during an estimated period when cases may have been reported (6-30 September) were scrutinised for their plasmid profile and potential exposures.All Salmonella reports to the local unit during this period as well as some before and after this period were reviewed for an epidemiological link to the implicated source.
All reported cases who fulfilled the definition of a probable or confirmed case (n=66) were interviewed using a standardised questionnaire on food consumption and other risk exposures, which was tailored to this outbreak (based, for example, on the restaurant menu).
The questionnaire was administered via telephone, or in person.The prototype had been developed, piloted and tested for validity in other outbreaks prior to this incident.All cases were interviewed within a week of exposure.The data were entered into a secure database, cleaned and cross-checked for inconsistencies.These case exposure data were used for descriptive analysis as well as the two analytical approaches described below.

Microbiology
Stool samples were available for all cases fulfilling the confirmed case definition (n=31) and from three of the four food handlers who worked in the takeaway restaurant.The food handlers were asymptomatic but were sampled to ensure that they were not the current or a continued source of the outbreak.Samples were initially cultured for Salmonella spp., Campylobacter spp., Escherichia coli or Shigella spp.Positive stool samples for Salmonella ssp.were sent to the National Salmonella Reference Laboratory, Health Protection Agency (now Public Health England) Centre for Infections, for phagetyping [12] and plasmid profiling [13] and tested for resistance to a range of antimicrobials by a standard breakpoint method.
During the investigation by the environmental health team on 16 September, a range of food and environmental samples were obtained and submitted for testing.A total of 30 food samples were taken from the implicated takeaway restaurant, and all raw shell eggs (n=72) from the premises were tested for Salmonella spp.Twelve environmental samples at the premises as well as leftover food samples from the household of an affected individual were cultured and characterised as above.

Data analysis
All the takeaway restaurant sales during the period at risk of exposure were entered into a cash register machine -regardless of whether payment was by cash or card.This machine automatically registers time and date; the price and product specification were entered manually by staff at the restaurant.Complete till receipt print-outs for the entire period where customers could have been at risk of exposure from contaminated food at the shop (two days prior to the first case until shop closure, 7-16 September 2009, inclusive)

Box
Case definitions, outbreak of Salmonella Enteritidis phage type 14b related to a north west London takeaway restaurant, United Kingdom, September 2009 A probable case was defined as a person developing diarrhoea or any two or more of the following symptoms: vomiting, fever (≥38°C) or abdominal pain within 72 hours of consumption of products from the implicated takeaway restaurant purchased between 6 and 16 September 2009.
A confirmed case was defined as a symptomatic person (see above) with a laboratory-confirmed isolate of S. Enteritidis phage type (PT)14b with resistance to nalidixic acid and with reduced susceptibility to ciprofloxacin from a blood or stool specimen.
Cases that travelled abroad in the seven days prior to symptom onset and cases with household contact to persons with diarrhoea or vomiting not associated with this outbreak were excluded.
were manually entered in an Excel spreadsheet, crosschecked and cleaned, and used for further analysis.
Till receipts are used for tax purposes and therefore precisely record the sales.Recording was accurate for date, time and price, but in this outlet, 25% of sales were recorded as miscellaneous.Information from the till receipts was used to reconstruct the population at risk, and the exposures in this population at risk for the comparative analysis and to generate sale volumes over time for the ecological case-crossover design.

Method 1 -a comparative approach
A theoretical cohort defining the population at risk, buying food from the restaurant between 7 and 16 September was constructed using sales information from the till receipts and estimated daily customer numbers from the restaurant owner.This was to establish a baseline sales' pattern by estimating the number of persons through the quantity and combination of individual food items bought.Individual food portions consumed by the cohort were calculated from sales, making assumptions about portion sizes and dish combinations.In principle the sale of one main dish (e.g.chicken kebab) or half a rotisserie chicken was equivalent to one portion.Side dishes (e.g.chips), sauces and sundries were counted as individual portions, if they were sold in the absence of a main dish.A person was assumed to consume one main dish (irrespective of an accompanying sundry) or one or more side dishes (in the absence of a main dish).It was assumed that joint food exposures in the cases were generalisable to the whole cohort.Miscellaneous food items were assumed to occur randomly by staff, person, and food product and have been excluded for the comparative analysis.The derived number of customers was compared with the information from the restaurant owner.
This data set with estimated numbers of individuals in the cohort consuming each food item was merged with the collected exposure information from the questionnaires administered to the cases.The theoretical exposures in the cohort were compared to the exposures in the cases providing estimated odds ratios and chi-squared tests of association.Each exposure was assessed only using single variable analysis.No multivariable analysis was attempted as the underlying assumptions preclude precise knowledge of joint exposure.

Method 2 -an ecological case-crossover analysis
The case-crossover design compares hypothesised risk exposures in the same individual in the interval before onset of illness to one or more intervals when the event does not occur [14].It is particularly suitable for continuous common source outbreaks [15], where unbiased exposure information (e.g. a menu or purchase records) is available [6,15].This analysis was performed to assess the a priori hypotheses that rotisserie chicken was the causal food source.This hypothesis was developed from customer complaint information, site visits, descriptive analysis and microbiological findings in a left-over piece of rotisserie chicken.
The ecological case-crossover design utilises the complete time series during the study period of sale volumes of rotisserie chicken, whereby the volume of sales in a time interval of one hour before the time of consumption as declared by the case (exposure interval), is compared to the sale volumes in the same hour in the preceding and following day (control intervals).
The difference in sales volumes between the exposure interval and the mean of the control intervals was assessed using a paired t-test.
In addition, a conditional logistic regression analysis was used to estimate the risk of illness per additional quarter chicken sold per hour.
All data were entered in EpiData 3.1 and statistical analysis was carried out using STATA SE 11.

Descriptive epidemiology
We identified 72 epidemiologically-linked cases with enteric illness in total, however three of these did not fulfill the clinical case definition and for a further three, not enough information was available to determine whether they fulfilled the clinical case definition.The remaining 66 cases of enteric illness therefore fulfilled the definition for probable or confirmed cases.Thirtyfive of the cases (53%) became ill on a single day (Figure 1).Most cases were male (41, 62%), and under 40 years of age (56, 85%).The mean age was 28 years (median: 26; range: 4-72).The majority of cases (42, 64%) resided near the takeaway restaurant.

Microbiology
Stool samples were available for 31 of the 66 cases; in all of them S. Enteritidis PT14b was isolated.Plasmid profiling was performed for seven of these isolates, and the profile was indistinguishable amongst them, but distinguishable by an additional 4 kb plasmid from profiles identified in other concurrent S. Enteritidis PT14b outbreaks in England and Wales.
All stool samples obtained from food handling staff at the venue (n=3) were negative for Salmonella.A food sample taken from a cooked leftover rotisserie chicken kept in the refrigerator of one of the cases and purchased at the implicated takeaway restaurant was a Based on observed data from the case questionnaires.
b The sum of portions in this column exceeds the total number in the theoretical cohort (n=2,390) based on the till receipt data, as persons may choose more than one dish.The numbers of portions are calculated for the outbreak period from 7 to 16 September 2009.c These are estimated exposures in the theoretical cohort using till receipt data and assuming that joint food exposures in the cases were generalisable to the whole cohort.
positive for S. Enteritidis PT14b.Environmental swabs taken from the floor of the refrigerator in the food preparation area and from a light switch in the staff toilet were positive for S. Enteritidis PT14b.The plasmid profile of the food sample and the environmental samples was indistinguishable from the organism isolated from the stools and the antimicrobial resistance profile was identical in all tested samples.All other samples including 12 environmental, 30 food samples and 72 egg shells from the implicated takeaway were negative for Salmonella.
The inspection by the environmental health team raised concerns about food preparation processes, such as the proximity of raw and cooked foods.In addition, an insufficient recording of temperature control, particularly for refrigeration and the rotisserie operation was noted.

Comparative analysis
Individual portions were estimated from the number of sales.An estimated 2,390 portions were sold during the ten days of the study period (7-16 September), a mean of 239 portions per day (range: 129-287).This broadly agreed with information from the owner, who estimated around 200 customers per day.25% of all sales were coded miscellaneous.
Table 1 provides an overview of the comparison between exposure information from the cases and estimated exposure in the theoretical cohort from the sales data, ordered by the number of explainable cases through the respective exposure.There was a significant association between consumption of rotisserie chicken and becoming a case in this analysis (odds ratio (OR): 2.75; confidence interval (CI): 1.67-4.52).Although similar or larger effect sizes were observed with other food items, consumption of rotisserie chicken can explain the majority of cases.It is possible that cross-contamination might explain some of these observations of large effect sizes, but which were affecting only few people.

Ecological case-crossover analysis
The times and volumes of rotisserie chicken sales corresponded well with the exposure times of cases (Figure 2), and based on this we formally analysed the correlation between rotisserie chicken sales and illness periods.

Discussion
We present the results of the investigation into a large outbreak associated with a north west London takeaway restaurant, using till receipts as a feasible, new analytical approach to validate the hypothesised causal exposure in this outbreak.While acknowledging the limitations of sales data, our analyses of till receipts provided epidemiological evidence of an association between the consumption of rotisserie chicken and developing a gastrointestinal illness.These findings were supported by microbiological and environmental findings.The ecological case-crossover study demonstrated that a linear relationship between chicken sales and risk of becoming a case occurred as soon as sales exceeded a certain amount.This may indicate that sales pressures led to substandard food handling practices, such as undercooking meat and cross-contamination.It was observed by some cases that raw chickens were being placed above roasted ones in the rotisserie and therefore potential cross-contamination through drippings could have occurred.Prior to serving, the chicken quarters were seared again on a hot grill before serving, in a rushed period this process may be inadequate, which may have caused the outbreak.This is useful information to change practices so as to prevent future outbreaks, reinforcing the need for scrupulous food hygiene practices at times of high demand.
Few published studies report the use of sales data in outbreak investigations.One of the reasons for this could be that access to sales data is not always granted to investigators, and our study benefits from the voluntary provision of complete till receipts by the restaurant owner.Supermarket bonus cards provide data on grocery shopping habits and have been used to generate hypotheses to inform subsequent analytical studies [10,16].Purchase records from supermarkets have also been used in a virtual cohort approach [17].The latter approach has some similarities to our study.Product purchase does not infer consumption, and in this study the unit of analysis was a group of people, which included a primary case (case-purchasing unit) [17].
Bonus card data allow identification of cases within a cohort, yield demographic information and thus allow combination with traditional methodologies.However, the use of bonus card schemes to investigate outbreaks is setting-specific, and limited by their popularity, representativeness and local data-sharing arrangements.
The best approximation of this data type for a small caterer was the use of till receipts, which contrary to bonus cards contained all sales during the period at risk.
Synnott et al. calculated the probability that frequencies of observed case kebab consumption were within the expected range of owner-reported sales frequencies in support of their case-control investigation in a small kebab shop outbreak [11].The analysis was carried out on a single food item, which all cases had been exposed to.In contrast, our study used a comparative approach for all possible exposures in the cohort, providing the exposures in the population at risk.
Our study benefits from extensive case-finding and good quality case exposure information acquired by applying a standardised exposure questionnaire in a standardised way.Till receipts contain readily available data, which are cheap, quick, accessible, and of good quality.Because till receipts are used for taxation purposes, data on price, date and time and food item (where entered) are likely to be accurate and unbiased.However, similar to other outbreak investigations, there remains potential for recall bias arising from the food exposure enquiry of the cases.
The main limitation of using till receipt data stems from the fact that sales data is not direct exposure/ consumption data, and that sales data do not allow an accurate calculation of a denominator.This is particularly relevant for the calculation of food exposures in

Figure 2
Descriptive plot of the relationship between quantity of chicken quarters sold and number of cases, outbreak of Salmonella Enteritidis phage type 14b related to a north west London takeaway restaurant, United Kingdom, 7-16 September 2009 The lines and left y axis denote the number of quarters of rotisserie chicken sold per time (5 minute intervals) and the red dots and right y axis denote the number of cases who were exposed to food from the takeaway restaurant at these times.
The figure illustrates that most cases (red dots) occurred shortly after sales peaks.the cohort of takeaway restaurant customers for direct comparison.Assumptions on portion sizes and dish combinations were wide ranging, and sales descriptions were not available for 25% of the sales (miscellaneous category).Future studies using this approach may wish to include 'sensitivity' analyses and vary the underlying assumptions.No information on illness or demography of all the customers was available and cases were included within the cohort.A further limitation is the inability to provide 'adjusted' associations to account for potential confounders due to lack of knowledge of the joint food exposures of the total cohort.It is possible to extrapolate from the joint food exposures in the cases and in the situation here, where the outbreak is likely to have a single causal exposure, the assumption that joint food exposures in the cases are generalisable to the whole cohort is realistic.
The case-crossover study is less vulnerable to these assumptions, and using exposures occurring immediately prior to when an individual case consumed food, and as this is compared to exposures at the same time of day, they act as their own controls, minimising the potential for selection bias [14,15].Confounding by subject characteristics is controlled by design [15].Information bias were largely avoided by use of purchase information [6], but there is a possibility that chicken sales were misclassified as 'miscellaneous' at busy times, although we were able to identify and recode a number of these on the basis of their typical price.While this would not change the direction of observed associations it could have led to an underestimate of the presented effect size.
However; this approach is more complex in terms of exposure data, potentially limiting the number of exposures that can be analysed.An a priori hypothesis is always required before commencing an analytical study, and is crucial in a case-crossover study.Results can be biased if control periods are not representative The figure demonstrates that the risk of becoming a case was correlated with increasing rotisserie chicken sales in the hour before. of the expected distribution of exposure for follow-up times that do not result in a case [6,14].The latter is usually unknown, but it is unlikely that the exposure changed systematically during the risk time [6] (same chicken batch) and we minimised this potential by taking the mean of two control periods.
In conclusion we demonstrated that the use of till receipt data for analytical outbreak investigation was feasible and provided additional evidence of an association between the consumption of rotisserie chicken purchased in a specific London takeaway restaurant and subsequent development of gastrointestinal illness.Our approach will need validation and refinement; however, understanding its limitations, the use of till receipt data provides additional information in outbreaks investigated with traditional methods, and is a practical approach to estimate exposures in outbreak investigations, where classical study designs are not feasible due to practical difficulties.

Figure 3
Figure 3Phylogenetic analysis with sequences from reference viruses downloaded from NCBI GenBank and GISAID EpiFlu databases of influenza B sequences derived from patients in the United Kingdom, 2012/13 influenza season

Figure 2
Figure 2Spectrum of single-nucleotide substitutions at the first nucleotide of the triplet coding for neuraminidase residue 155 of the influenza A(H1N1)pdm09 viruses with Y155H mutation, Spain, 2012/13 (n=2)

Figure 1
Figure 1Epidemic curve obtained from investigating an outbreak of Salmonella Enteritidis phage type 14b related to a north west London takeaway restaurant, United Kingdom, September 2009 (n=66 cases a )

Table 1
Reference influenza B haemagglutinin 1 sequences used in phylogenetic analysis

Table 2
Details for influenza A and B cases and controls, United Kingdom, October 2012-April 2013 (n=3,286 individuals) HPS: Health Protection Scotland; PHA: Public Health Agency; RCGP: Royal College of General Practitioners' surveillance scheme; SMN: PHE Specialist Microbiology Network.

Table 4
Adjusted vaccine effectiveness estimates for influenza by age, surveillance scheme and by time since vaccination, United Kingdom, October 2012-April 2013 CI: confidence interval; HPS: Health Protection Scotland; PHA: Public Health Agency; RCGP: Royal College of General Practitioners' surveillance scheme; SMN: Public Health England Specialist Microbiology Network, VE: vaccine effectiveness.a Adjusted for age group, sex, month of sample collection and surveillance scheme.b Number of vaccinated cases/controls too low to give an estimate with meaningful precision (95% CI lower end <-200 and upper end >80).c Unadjusted Cornfield 95% CI.

Table 3
Adjusted vaccine effectiveness estimates based on samples positive (cases, N=1,330) and negative (controls, N=1,956) for influenza according to vaccination status, United Kingdom, October 2012-April 2013 Because two individuals positive for influenza A(H3N2) and B, and one individual positive for A(H1N1)pdm09 and B are included in both the influenza B and influenza A cases, summing up the cases presented in this column amounts to 1,333 instead of 1,330.b Adjusted for age group, sex, month of sample collection and surveillance scheme. a

Table 2
Neuraminidase inhibition of influenza A(H1N1)pdm09 virus isolates, Spain, 2012/13 (n=2) Data were obtained from two independent MUNANA assays perfomed at the National Influenza Centre of Madrid, against the ISIRV reference virus panel.Each virus was assayed in duplicate.Data are expressed as mean IC50 values in nM.WT denotes isolates not containing any NA substitution related to reduced inhibition by NAIs.

Table 1
Results of the comparative till receipt analysis, outbreak of Salmonella Enteritidis phage type 14b related to a north west London takeaway restaurant, United Kingdom, September 2009The table is ordered by number of cases explainable by respective exposures (i.e. the numbers of cases who reported the particular exposure, column 1).
On average, 34.59 quarters of chicken corresponding to 8.6 chickens (CI: 6.8-10.5)weresold in the hour before the cases had consumed food from the takeaway.This compares with an average of 19.75 quarters, or 4.9 chickens (CI: 4.4-5.5)sales, in the two reference periods.The difference of 14.89 quarters, i.e. 3.7 chickens (CI: 2.2-5.2) is significantly different from zero (p <0.0001) (Table2).The clustering of cases around the highest sales peaks, particularly on 10 September is shown in Figure2.

Table 2
Results of the case-crossover analysis, outbreak of Salmonella Enteritidis phage type 14b related to a north west London takeaway restaurant, United Kingdom, September 2009 Differences between chicken sales in exposed and non-exposed times were compared with t tests.bThelower part shows the result of the conditional logistic regression model estimating the risk of illness per chicken quarter sold per hour. a