Vaccine 37 (2019) 2470–2476 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Molecular influenza surveillance at a tertiary university hospital during four consecutive seasons (2012–2016) in Catalonia, Spain Cristina Andrés a, Paula Peremiquel-Trillas b, Laura Gimferrer a, Maria Piñana a, Maria Gema Codina a, José Ángel Rodrigo-Pendás b, Magda Campins-Martí b, María Carmen Martín a, Francisco Fuentes a, Susana Rubio a, Tomàs Pumarola a,⇑, Andrés Antón a a Respiratory Viruses Unit, Virology Section, Microbiology Department, Hospital Universitari Vall d’Hebron, Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain b Preventive Medicine and Epidemiology Department, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Vall Hebron Research Institute, Barcelona, Spain a r t i c l e i n f o a b s t r a c t Article history: Background: Influenza viruses (FLUV) are continuously evolving, which explain the occurrence of sea- Received 2 August 2018 sonal influenza epidemics and the need to review the vaccine strain composition annually. The aim is Received in revised form 19 March 2019 to describe the genetic diversity and clinical outcomes of FLUV detected at a tertiary university hospital Accepted 21 March 2019 in Barcelona (Spain) during the 2012–2016 seasons. Available online 27 March 2019 Methods: The detection of FLUV from patients attended at the Emergency Department or admitted to the hospital was performed by either immunofluorescence or PCR-based assays. A specific real-time one-step Keywords: multiplex RT-PCR was performed for influenza A (FLUAV) subtyping. The complete coding haemagglu- Influenza viruses Surveillance tinin domain 1 (HA1) and neuraminidase (NA) (2015–2016) protein sequences from a representative Respiratory tract infection sampling were molecular characterised. Genetic diversity Results: A total 1774 (66.1%) FLUAV and 910 (33.9%) influenza B (FLUBV) cases were laboratory- Molecular epidemiology confirmed. The hospitalisation rate was different between seasons, being the highest (81.4%) during the 2014–2015 season. FLUV were genetically close to vaccine strains except to the 2014–2015, in which most characterised A(H3N2) viruses belonged to a genetic group different from the vaccine strain. During the 2015–2016 season, B/Victoria-like viruses were the most predominant, but this component was not included in the trivalent vaccine used. Mutations D222G or D222N in HA1-domain were found in 3 A (H1N1)pdm09 strains from ICU-admitted cases. Three A(H1N1)pdm09 strains carried the NA H275Y (2) and S247N (1) mutations, respectively related to resistance or decreased susceptibility to oseltamivir. Conclusions: The circulation of drifted A(H3N2) strains during the 2014–2015 season was related to the high hospitalisation rate due to the mismatch with the vaccine strains. The predominance of a FLUBV lin- eage not included in the trivalent influenza vaccine during the 2015–2016 season highlights the need to use a tetravalent influenza vaccine. Virological surveillance of viral variants carrying protein changes that alter tropism and susceptibility to antivirals features should be strengthened in hospital settings. Ó 2019 Elsevier Ltd. All rights reserved. 1. Background FLUV are single-stranded, negative-sense, segmented RNA viruses belonging to the Orthomyxoviridae family [3,4]. Based on Influenza viruses (FLUV), one of the main causative agents of genetic and antigenic features, three different types of human respiratory infections, are related to high morbidity and mortality FLUV (A, B and C) are currently described. According to antigenic in the community, mainly in high-risk patients such as the elderly, properties of the surface proteins, haemagglutinin (HA) and neu- those with underlying comorbidities or pregnant women [1,2]. raminidase (NA), influenza A viruses (FLUAV) are divided into dif- ferent subtypes (H1N1pdm09 and H3N2) [3,5], as well as influenza B viruses (FLUBV) into two major lineages, B/Victoria/2/87-like ⇑ Corresponding author at: Respiratory Viruses Unit, Virology Section, Microbi- (FLUBV/VIC) and B/Yamagata/16/88-like (FLUBV/YAM) [6]. ology Department, Hospital Universitari Vall d’Hebron, Vall d’Hebron Research FLUV are continuously evolving through amino acid substitu- Institute, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron 119-129, tions altering antigenic properties (antigenic drift) or, less fre- 08035 Barcelona, Spain. quently, by segment reassortment events (antigenic shift) [7]. E-mail address: virusrespiratoris@vhebron.net (T. Pumarola). https://doi.org/10.1016/j.vaccine.2019.03.046 0264-410X/Ó 2019 Elsevier Ltd. All rights reserved. C. Andrés et al. / Vaccine 37 (2019) 2470–2476 2471 Some amino acid substitutions in the HA receptor binding site 2.3. Detection of respiratory viruses (RBS) can alter the cellular host tropism, conditioning the species range and tissue tropism, which may be also related to viral viru- The detection of FLUV and other respiratory viruses (respiratory lence [8]. The high mutation rate of the NA protein can also affect syncytial virus, metapneumovirus, adenovirus, rhinoviruses, enter- the antiviral susceptibility to currently available neuraminidase oviruses, coronaviruses, parainfluenza viruses and bocavirus) was inhibitors (NAIs) drugs [9]. In addition, changes in the antigenic carried out by either immunofluorescence antigen detection features of FLUAV or FLUBV, mainly in HA, explain the occurrence (SOFIA Influenza A + B, Quidel, California, USA; and D3 Ultra 8TM of seasonal influenza epidemics and are the reason for the annual DFA Respiratory Virus Screening & Identification Kit Diagnostic vaccine composition update needed for a more effective preven- HYBRIDS, USA) or PCR-based assays (Anyplex II RV16 Detection tion of disease and the related complications in high-risk patients Kit, Seegene, Korea; and, GeneXpert Flu, Cepheid, USA). Prior to [10]. Vaccination is the most effective public health action for pre- PCR-based assays, total nucleic acids were extracted using Nucli- venting influenza. A higher vaccination enhances the achievement Sens easyMAG (bioMérieux, Marcy l’Etoile, France) according to of community immunisation [11]. In Catalonia, the influenza vacci- the manufacturer’s instructions, and kept frozen ( 20 °C) until nation is recommended to individuals over 60 years of age, espe- use. An in-house real-time one-step multiplex RT-PCR [15] was cially those living in nursing homes, children over 6 months of performed for HA subtyping of seasonal influenza A viruses age and adults with high risk comorbidities for clinical complica- (H1pdm09 and H3). tions due to influenza infection, such as pregnancy or chronic dis- eases. It is also recommended to healthcare professionals and 2.4. Amplification and sequencing of influenza haemagglutinin and people working for public services (police, firefighters or teachers). neuraminidase genes The influenza vaccines available in Catalonia are subunit, adju- vanted and cell-based vaccines. The tetravalent vaccines have only The complete coding region of HA1-domain of a representative been available since the 2016–2017 season for the vaccination of number of influenza laboratory-confirmed specimens from hospi- high-risk patients at hospital settings [12]. The vaccination cover- talised patients was sequenced for phylogenetic analysis and age in our region is variable and goes from 10 to 70% depending on molecular characterisation. In addition, during the 2015–2016 sea- the age group, although the mean coverage is around 35% each sea- son, the complete NA coding region was also sequenced for the son [13]. screening of amino acid substitutions associated with a reduced The aim of this study is to describe the genetic features of antiviral susceptibility [9,16]. HA and NA amplifications were car- detected viruses and the clinical outcome of influenza cases ried out by a one-step RT-PCR assay using the one-step RT-PCR Kit attended at Vall d’Hebron University Hospital (Barcelona, Spain) (Qiagen, Hilden, Germany) with the primers and PCR protocols from the 2012–2013 to the 2015–2016 seasons. shown in Supplementary Table 1. PCR products purification was subsequently performed using Exo-SAP-IT (USB, Affymetrix Inc. Cleveland, Ohio, USA) and sequenced by the ABI Prism Big Dye Ter- 2. Material and methods minator cycle sequencing kit v3.1 (Thermo Fisher Scientific, Wal- tham, MA, USA) on the ABI PRISM 3130XL Genetic Analyzer 2.1. Sampling (Thermo Fisher Scientific, Waltham, MA, USA) with the sequencing primers (Supplementary Table 1). Nucleotide sequences were edi- Vall d’Hebron University Hospital is the largest tertiary hospital ted and assembled using SeqScape v2.6 software (Thermo Fisher in Catalonia and the second one in Spain, covering more than Scientific, Waltham, MA, USA). Sequences of the present study 450.000 inhabitants. From October 2012 (epidemiological week were submitted to the Global Initiative on Sharing Avian Influenza 40/2012) to May 2016 (epidemiological week 20/2016), upper Data (GISAID available at www.platform.gisaid.org). (nasopharyngeal aspirates or swabs) and lower (bronchoalveolar lavages, bronchoaspirates or tracheal aspirates) respiratory tract 2.5. Detection of polymorphisms and phylogenetic analysis specimens were collected for influenza and other respiratory viruses laboratory-confirmation from patients with suspicion of Phylogenetic analyses of complete HA1-domain coding region acute respiratory tract infection who were attended at the Emer- sequences were performed using the reference sequences by sub- gency Department that accomplished the criteria to be admitted type (FLUAV) or by lineage (FLUBV) by season in the Northern to the hospital; from patients that could benefit from antiviral Hemisphere recommended by the European Centre for Disease treatment without needing hospitalisation; or from hospitalised Prevention and Control (ECDC) (Supplementary Table 2). Both mul- patients that acquired influenza nosocomial infection. Based on tiple nucleotide sequence alignment using the MUSCLE algorithm medical records, some demographic features (age and sex), hospi- [17] and the molecular evolutionary model analysis were con- talisation, Intensive Care Unit (ICU) admission, outcome and the ducted in MEGA v5.2 [18]. The phylogenetic trees were constructed administration of antiviral treatment were retrospectively col- using a neighbor-joining (NJ) distance method as implemented in lected from influenza laboratory-confirmed cases. MEGA v5.2 with the nucleotide substitution model with the lowest Bayesian information criterion (BIC) score [18]. The topological accuracy of the internal branch was evaluated by the bootstrap 2.2. Definition of SARI cases method (1000 replicates). In order to detect amino acid substitutions in the complete According to the Catalan Public Health Agency, a severe hospi- HA1-domain and NA coding regions, the deduced amino acid talised patient is described as that case with pneumonia due to sequences from aligned nucleotide sequences were compared to influenza virus or to bacterial infection, septic shock, multiorganic those of the vaccine strains recommended by the World Health failure, acute respiratory distress or admission to Intensive Care Unit (ICU). Moreover, antiviral (NAIs) treatment was administered Organisation (WHO) (A(H1N1)pdm09: A/California/07/2009 when influenza infection was laboratory-confirmed for those (2012–2016); A(H3N2): A/Victoria/361/2011 (2012–2013); A/Tex- patients who are hospitalised due to the severity of the disease as/50/2012 (2013–2015); and A/Switzerland/9715293/2014 or belonging to high-risk groups [14] to prevent further disease (2015–2016); B/Yamagata: B/Wisconsin/01/2010 (2012–2013); B/ complications. Massachusetts/02/2012 (2013–2015); and B/Phuket/3073/2013 2472 C. Andrés et al. / Vaccine 37 (2019) 2470–2476 (2015–2016); B/Victoria: B/Brisbane/60/2008 (2012–2016)). Pri- the hospitalisation rate was different between seasons mers and protocols are available under request. (p < 0.001), being the highest (81.4%) during the 2014–2015 sea- son. The hospitalisation rates were also significantly different between influenza types (FLUAV: 60.8% vs. FLUBV: 45.6%; 2.6. Statistical analysis p < 0.001) or FLUAV subtypes (H1N1pdm09: 55.9% vs. H3N2: 64.6%; p < 0.001). Among hospitalised patients, ICU-admissions Statistical analysis was performed using STATA v14 (StataCorp. were also different by season throughout the study period and 2015 Stata Statistical Software: Release 14. College Station, TX: showed a decreasing trend (p < 0.001). While no differences on StataCorp LP). A descriptive analysis was performed, calculating ICU admissions between influenza types (FLUAV: 3.5% vs. FLUBV: the median as a measure of central tendency as well as the 2.6%; p = 0.444) were observed, these were different between interquartile range as a measure of its dispersion. Categorical FLUAV subtypes (A(H1N1)pdm09: 3.9% vs. A(H3N2): 2.2%; variables were described through frequencies and proportions. p = 0.037). Through the study period, there was also a decreasing Chi-squared test was calculated to assess associations between trend in mortality (p = 0.003), but no differences were observed categorical variables. P values < 0.05 were considered to be between influenza types (p = 0.116) or FLUAV subtypes statistically significant. (p = 0.572). Nevertheless, the high number of fatal cases attributed to A(H1N1)pdm09 during the 2012–2013 season was remarkable 3. Results (7.8%) in comparison with later seasons. Phylogenetic analyses of HA1 sequences revealed the high A total of 18,405 specimens from 12,680 cases (median age: genetic diversity of FLUV, distinguishing different genetic clades 14.5 years; female: 46%) were received for respiratory viruses’ and subclades, represented in Fig. 2(A–D) and Supplementary laboratory-confirmation, of which 2866 (16%) specimens from Table 3. Regarding A(H1N1)pdm09 viruses, all sequences (2 6 8) 2684 (21%) patients (median age: 36 years; female: 49%) were clustered into clades 6 and 7 during the 2012–2013 season. During laboratory-confirmed for 1774 (66.1%) FLUAV (female: 48.4%) the following seasons, sequences fell into two subgroups (6B.1 and and 910 (33.9%) FLUBV (female: 50.2%). FLUV detection was usu- 6B.2) within clade 6B (Fig. 2A), in which the strains (6B.1) gained a ally reported in winter months, and only a few sporadic cases were new glycosylation site (S162N) [19]. In addition, all A(H3N2) detected during the interseason periods (Fig. 1). In addition, detec- sequences (386) but one clustered within genetic clade 3 tion of FLUV steadily increased during the study period (p < 0.001). (A/Slovenia/537/2011-like), in different genetic subgroups (3C.2a, A variable co-circulation of the different influenza types was A/HongKong/4801/2014-like; 3C.3, A/Samara/73/2013-like; and shown, since FLUAV circulated during all seasons, while FLUBV 3C.3a, A/Switzerland/9715293/2013-like) (Fig. 2B). However, did not circulate during the 2013–2014 season. Moreover, a partic- during three seasons the detected strains were closely related to ular influenza type or subtype was also predominant in each stud- the included vaccine strains, while during the 2014–2015 ied season: FLUBV during the 2012–2013 season; A(H3N2) during season A(H3N2) viruses belonged to the 3C.2a genetic subset the 2013–2014 and 2014–2015 seasons; and A(H1N1)pdm09 and (A/HongKong/4801/2014-like) that showed different antigenic FLUBV during the 2015–2016 season, as shown in Fig. 1. features from vaccine strain included that season according to Data regarding demographic features (age and sex), hospitalisa- WHO/ECDC data [20]. Furthermore, during the 2015–2016 season tion rate, ICU-admission, antiviral treatment administration and there was a shift in the predominance of FLUBV lineage to fatal outcomes of influenza-confirmed cases by season are sum- FLUBV/VIC (Fig. 2C) (57 sequences), which was not included in marised in Table 1. Overall, the median age of FLUAV-confirmed the trivalent vaccine, since during the previous seasons, all FLUBV cases (44 years; IQR 5–70 years) was significantly higher viruses belonged to FLUBV/YAM lineage (Fig. 2D). FLUBV/YAM (p < 0.001) than that of FLUBV-confirmed cases (23 years; IQR 4– viruses (134) detected during the 2012–2013 season belonged 62 years). However, no significant differences (p = 0.489) between mostly to clade 2 (B/Massachusetts/02/2012-like) and only a few A(H1N1)pdm09 (42 years; IQR 3–67 years) and A(H3N2) (46 years; to clade 3 (B/Phuket/3073/2013-like), while all FLUBV/YAM IQR 7–74 years) were found. Though, differences in the distribution detected during 2014–2015 corresponded to clade 3 (B/Phuket/ by age groups were found between influenza types (p < 0.001) or 3073/2013-like). subtypes (p < 0.001). The characterisation of amino acid sequences of coding HA1- The hospitalisation rate for influenza laboratory-confirmed domain sequences also revealed the detection of some viral vari- cases was overall 55.7% (1494/2684 cases), with a balanced ants carrying the following amino acid substitutions in the RBS male:female proportion (female: 49.0%; p = 0.398). Furthermore, [21,22]: S137F (1 case), I192S (1 case) and I192T (2 cases) among Fig. 1. Distribution of Severe Acute Respiratory Infections (SARI) cases and the positive cases for Influenza and other respiratory viruses detected from the epidemiological weeks 40/2012 to 20/2016. The period of winter in the North Hemisphere is highlighted in blue. Detections were performed by either immunofluorescence assay or real time RT-PCR. The period of influenza vaccine campaign begins in October (epidemiological week 42) in Catalonia (Spain). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) C. Andrés et al. / Vaccine 37 (2019) 2470–2476 2473 A(H3N2) viruses; and P241S (1 case) among FLUBV, all found in Numbers may not add due to the existence of Influenza A viruses not subtyped. Season 2012–2013: 5 (10,2%); season 2013–2014: 48 (12,1%); season 2014–2015: 101 (1,4%); season 2015–2016: 55 (8,3%); global: 209 (11,8%). Detection, age (median), age categories/groups, female, hospitalisation, ICU-admission, antiviral treatment and mortality rates (N; %) by influenza types or subtypes and season (% in columns, depending on the influenza type/subtype). Numbers may not add due to the existence of Influenza A viruses not subtyped. Season 2012–2013: 2 (40,0%); season 2013–2014: 31 (64,5%); season 2014–2015: 89 (88,1%); season 2015–2016: 25 (45,5%); global: 147 (70,3%). p trend < 0.001 p trend = 0.003 Numbers may not add due to the existence of Influenza A viruses not subtyped. Season 2012–2013: 1 (20,0%); season 2013–2014: 6 (12,5%); season 2014–2015: 4 (3,9%); season 2015–2016: 2 (3,6%); global: 13 (6,2%). samples from the upper respiratory tract. In addition, D222G (1 Numbers may not add due to the existence of Influenza A viruses not subtyped. Season 2012–2013: 0 (0,0%); season 2013–2014: 4 (8,3%); season 2014–2015: 1 (1,0%); season 2015–2016: 1 (1,8%); global: 6 (2,9%). case) and D222N (2 cases) mutations of A(H1N1)pdm09 viruses p < 0.0001 p = 0.398 p = 0.116 p = 0.037 p < 0.001 p < 0.001 p-value during the 2012–2013 season but not later, were detected in upper (2 cases) and lower (1 case) respiratory specimens from three ICU- admitted patients, one of which with fatal outcome (D222N). These FLUBV 910 (33.9) mutations in the RBS were associated to severe disease in previous studies [23–26]. 285 (31.3) 212 (23.3) 415 (45.6) 140 (15.4) 457 (50.2) 98 (10.8) 175 (19) 24 (2.6) 14 (1.5) Regarding amino acid substitutions associated with reduced 23 NAI susceptibility [9,16], two mutations were found in three 2015–2016 A(H1N1)pdm09 viruses (H275Y: 2 cases; S247N: 1 916 (51.6) 649 (36.6) 352 (38.4) 198 (30.5) 420 (45.9) 325 (50.1) 512 (55.9) 419 (64.6) 257 (28.1) 240 (37.0) 156 (17.0) 92 (14.2) 73 (11.2) 47 (7.2) 14 (2.2) 16 (2.5) case) in respiratory samples collected from immunosuppressed FLUAV 1774 (66.1) patients, who favourably responded to a standard five-day oselta- H1pdm09 H3 46 mivir treatment. Samples from two cases (H275Y and S246N) were 2684 (14.6) 1494 (55.7) 1314 (49.0) 99 (10.8) 52 (5.7) 87 (3.2) 36 (3.9) 46 (1.7) 15 (1.6) collected before treatment, and the other one, one day after the 18,405 TOTAL onset of start of treatment. 42 FLUBV 563 (46.0) 4. Discussion 112 (19.9) 157 (27.9) 277 (49.2) 166 (29.5) 114 (20.2) 118 (21.0) 62 (11.0) 9 (1.6) 2 (0.4) In the present study, the prevalence, the genetic diversity and 13 the clinical outcomes of detected FLUV in patients attended or hos- 593 (89.7) 13 (2.0) 101 (17.0) 2 (15.4) 217 (36.6) 3 (23.1) 190 (32.0) 8 (61.5) 273 (46.0) 9 (69.2) 296 (49.9) 6 (46.2) 1 (7.7) 0 (0.0) 0 (0.0) 0 (0.0) pitalised at a tertiary hospital in Catalonia are reported, which FLUAV 661 (54.0) H1pdm09 H3 69 might be considered a continuation of the influenza surveillance 12235 (18.8) 2015–2016 589 (48.2) 493 (40.3) report from 2006 to 2012 in Catalonia previously published [15]. 53 (8.9) 32 (5.4) 28 (2.3) 17 (2.3) 13 (1.1) 9 (1.5) As shown in Fig. 1, the number of FLUV detected cases per sea- 6489 47 son increased over the season, which might be related to the improvement of the detection methods by a broader use of PCR- FLUBV 215 (24.3) based methods rather than antigen-detection based assays [27] 181 (84.2) 108 (50.2) in our laboratory, as well as the strengthened surveillance of respi- 88 (40.9) 86 (40.0) 17 (7.9) 15 (7.0) 9 (4.2) 3 (1.4) 2 (0.9) ratory viruses in our hospital. 56 The co-circulation of FLUAV and FLUBV during these four con- secutive seasons was highly variable, with shifts in the predomi- 394 (59.1) 105 (26.6) 179 (45.4) 188 (47.7) 307 (77.9) 47 (11.9) 39 (9.9) 25 (6.3) 12 (3.0) nance of influenza types and subtypes. Overall, the circulating 4 (1.0) (75.5) FLUV showed similar proportions to those reported in other Span- H3 58 ish regions [28], but showing some differences in comparison to 2014–2015 FLUAV 667 719 (81.4) 883 (17.0) 172 (25.8) 141 (82.0) H1pdm09 28 (16.3) 46 (26.7) 79 (45.9) 19 (11.0) 70 (40.7) 429 (49) 17 (1.9) 18 (2.0) other European countries [29]. Previous studies already reported 9 (5.2) 2 (1.2) 7 (5.0) During the season 2015–2016 one patient presented a co-infection between FLUAV and FLUBV. 5189 some differences between Catalonia and other Spanish regions 48 [15]. FLUAV was predominant in three out of the four studied sea- FLUBV 0 (0.0) sons, with the only exception of the 2012–2013 season when FLUBV was the most frequently detected, similarly to other Span- ish regions but different from other European countries [29]. At subtype level, during the 2013–2014 season, A(H3N2) was pre- 236 (59.0) 125 (53.0) 102 (43.2) 51 (21.6) 87 (36.9) 25 (10.6) 52 (22.0) dominantly detected in Catalonia based on these results and also 21 (8.9) 10 (4.2) 3 (1.3) (100) on data provided by the Public Health Agency [30], in contrast to H3 30 other Spanish communities and other European countries 2013–2014 FLUAV 397 397 (11.1) 185 (46.6) 210 (52.9) 113 (28.0) H1pdm09 18 (15.9) 18 (15.9) 20 (17.7) 50 (44.2) 60 (53.1) 52 (46.0) [28,29]. This heterogeneity and variability at temporal and geo- 23 (5.8) 7 (6.2) 7 (6.2) 8 (2.0) 1 (0.9) 3571 graphical level of FLUV circulation reinforce the idea to maintain 29 a local and continuous influenza surveillance. FLUBV 132 (72.9) The clinical impact of influenza epidemics in the community is always considered a valuable data to be reviewed, taking into (15.9) (14.4) 72 (54.5) 68 (70.1) (30.3) (30.3) account the FLUV genetic diversity. Therefore, some differences (9.1) 12 (9.1) 5 (3.8) were found between influenza types (A vs B) or subtypes 21 19 12 40 40 8 (H1pdm09 vs H3), or vice versa regarding the age and the clinical 38 (77.6) 6 (12.2) 1 (16.7) 1 (16.7) 1 (16.7) 23 (60.5) 4 (66.7) 3 (50.0) outcomes of influenza-confirmed cases, such as hospitalisation, 0 (0.0) 0 (0.0) 15 (39.5) 3 (50) FLUAV 49 (27.1) 38.5 ICU-admission, and mortality rates. In the present study, the pop- H1pdm09 H3 0 2012–2013 ulation susceptible to FLUAV infection seemed to be older than that 181 (5.7) 86 (47.5) 97 (53.6) 7 (18.4) 9 (23.7) 8 (21.1) 4 (10.5) 18 (9.9) 5 (13.0) susceptible for FLUBV, although FLUAV was predominant in almost 3 (7.9) 8 (4.4) 3 (8.0) 3156 all age groups, except to 5–14-year-old group in whom FLUBV pre- 9 vailed as previously reported by other authors [31,32]. On the Influenza subtype1 other hand, while no differences were found between A(H1N1) Received samples pdm09 and A(H3N2), the distribution of cases by age groups was ICU-admitted3 Influenza type Age (median) 15–64 years 2 5–14 years different. The hospitalisation rate was variable throughout the Inpatients 2–4 years >64 years <2 years Female Exitus4 study reporting differences between seasons, which might be Table 1 related to the predominant influenza types or subtypes. Overall, 1 2 3 4 5 FLUAV and A(H3N2) were related to higher hospitalisation rates 2474 C. Andrés et al. / Vaccine 37 (2019) 2470–2476 than FLUBV and A(H1N1)pdm09, respectively. However, the asso- However, despite antigenic stability, A(H1N1)pdm09 is still caus- ciation of A(H3N2) with a higher hospitalisation and lower ICU- ing severe hospitalisations. admission rates than A(H1N1)pdm09 might be explained due to On the other hand, A(H3N2) virus has shown a great capability the high number of hospitalised cases during the 2014–2015 sea- to evolve quickly due to maybe the immune pressure from the son when a drifted A(H3N2) virus was circulating [20]. Mortality human population infected because it has been globally circulat- rate was declining along the study period, not showing differences ing for more than 50 years. During the last seasons, A(H3N2) between FLUV types or subtypes, but highlighting the high mortal- viruses acquired a great number of amino acid substitutions, ity rate for A(H1N1)pdm09 during the 2012–2013 season when a diverging in different genetic clades and subclades as shown in low number of FLUAV cases was reported. This decreasing mortal- the present study, and continuously changing the potential glyco- ity rate over the four seasons could be related to the increase fre- sylation features of HA. In fact, as previously commented, the quency of sampling due to the introduction of PCR-based high activity of A(H3N2) viruses during the 2014–2015 season methods to speed diagnostic and treatment and reducing was related to the circulation of A(H3N2) viruses antigenically complications. different from the vaccine strain used [20]. However, the unpre- Based on phylogenetic analysis of HA1 sequences, A(H3N2) dictable evolution of FLUV together with the current challenges showed a higher heterogenicity than A(H1N1)pdm09. Despite the for the correct antigenic characterisation of A(H3N2) viruses, emergence, the spread and the genetic divergence of A(H1N1) make difficult to forecast the vaccine composition for upcoming pdm09 strains into different genetic subgroups (6B.1 and 6B.2) seasons [20]. within A(H1N1)pdm09 6B clade, as reported worldwide [33], the Regarding FLUBV, there was co-circulation of both lineages, but current A(H1N1)pdm09 viruses remained antigenically similar to with the predominance of FLUBV/YAM in two out of three seasons the vaccine strain since the beginning of the pandemic [20,34]. with FLUBV detection reported as well as the no correspondence Fig. 2. (A–D): Phylogenetic trees of HA1-domain sequences from influenza A(H1N1)pdm09 (A), A(H3N2) (B), B/VIC (C) and B/YAM (D) strains. The Tamura-Nei (TN93) method with gamma-distribution was used to carry out the analysis. Almost all sequences corresponding to influenza viruses detected during the 2012–2016 season are compressed, although some of them are labelled in green. Sequences used as reference are labelled in red and seasonal vaccine strains, in blue and bold. Only those bootstrap values over 70% are shown. Amino acid substitutions that define every phylogenetic cluster are marked in purple at the nodes, underlined those related to antigenic sites, and derived from each reference sequence (A(H1N1)pdm09: A/California/07/2009, A(H3N2): A/Perth/16/2009, B/Yamagata: B/Florida/04/2006, B/Victoria: B/Malaysia/2506/2004). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) C. Andrés et al. / Vaccine 37 (2019) 2470–2476 2475 Fig. 2 (continued) with the strain lineage included in the trivalent vaccine composi- tion of severe influenza cases, since the virological surveillance of tion. The shift on the predominance of both lineages is well- influenza viruses is overall carried out from samples collected from known, and sometimes the circulating lineage is not included in community-based surveillance networks. On the other hand, the the trivalent vaccine, which highlights the need of using the emergence of genetically resistant variants to antiviral drugs is tetravalent influenza vaccine to give a better and wider protection one of the main concerns of influenza surveillance and Public to the high-risk population. Therefore, the vaccination with Health worldwide [9,16]. In the present study, three viruses carry- tetravalent vaccines in primary care would both improve the atti- ing the mutations H275Y and S247N, associated with resistance or tude towards influenza vaccination and have a beneficial effect on reduced susceptibility to NAIs, were found in respiratory population coverage. specimens from hospitalised patients under standard oseltamivir In addition to the circulation of both FLUBV lineages, FLUBV also treatment during the 2015–2016 season. The fact that immuno- showed a continuous evolution based on HA sequences. Therefore, suppression status and antiviral treatment administration are con- FLUBV/YAM viruses belonged to two genetically and antigenically sidered high-risk factors for the selection and emergence of divergent genetic clades (clade 2 and 3) [29], one of which (clade resistant strains [35], conditions that are accomplished by many 3) was not represented by the corresponding vaccine strain during of the patients hospitalised in tertiary centres, highlights the the 2012–2013 season. importance to strengthen the virological surveillance of influenza In regard to the detection of virulence and resistance markers, A viruses in hospital settings. While surveillance tasks in (H1N1)pdm09 has the capability to acquire mutations that can community-based networks is highly valuable, the information alter the tropism or the antiviral susceptibility during the infection reported in hospital settings, where cases of severe disease or [24,35]. On one hand, A(H1N1)pdm09 strains from ICU-admitted antiviral resistance are attended, should be always considered to cases carrying mutations D222G or D222N in HA1, which are still monitor circulating viruses, in particular those with novel pheno- the unique genetic markers related to disease severity [23–26], typic features. were detected during the 2012–2013 season but not in later sea- In summary, our results highlight the prevalence of FLUV in sons, when a higher A(H1N1)pdm09-related mortality rate was patients admitted to a tertiary hospital and its variable and unpre- reported. Surely, the prevalence of variants carrying these muta- dictable circulation. The capability of FLUV to acquire genetic tions might be underestimated due to a suboptimal characterisa- diversity that can affect viral features related to antigenicity, trop- 2476 C. Andrés et al. / Vaccine 37 (2019) 2470–2476 ism, and susceptibility to antivirals makes virological surveillance, [13] Sistemes d’Informació dels Serveis d’Atenció Primària (SISAP). Institut Català de la Salut (ICS). Available at: . hospital settings. [14] Pla d’actuació a Catalunya enfront d’una infecció per virus de la grip en fase post-pandèmica. Sub-direcció General de Vigilància i Resposta a Emergències de Salut Pública. Generalitat de Catalunya, Departament de Salut.; Gener 2018. Ethical approval Available at: . Institutional Review Board approval (PR(AG)329/2014) was [15] Antón A, Marcos MA, Torner N, Isanta R, Camps M, Martínez A, et al. Virological surveillance of influenza and other respiratory viruses during six consecutive obtained from the Vall d’Hebron University Hospital (HUVH) seasons from 2006 to 2012 in Catalonia, Spain. Clin Microbiol Infect 2016;22 Clinical Research Ethics Committee. (564):e1–9. [16] Nguyen HT, Fry AM, Gubareva LV. Neuraminidase inhibitor resistance in influenza viruses and laboratory testing methods. Antivir Ther Conflict of interest 2012;17:159–73. [17] Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high The authors have no conflicts of interest to declare. throughput. Nucl Acids Res 2004;32:1792–7. [18] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, Acknowledgements evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731–9. [19] World Health Organisation (WHO). Recommended composition of influenza This work has been partially supported by Fondo de Investi- virus vaccines for use in the 2016-2017 northern hemisphere influenza gación Sanitaria (grants FIS PI08/0118, FIS PI11/01864, FIS season; 2016. Available at: . [20] World Health Organisation (WHO). WHO recommended candidate viruses and Universities; Agència d’Avaluació de Tecnologia i Recerca potency testing reagents for the development and production of vaccines for Mèdiques (AATRM) (grant 402/02/2008); Catalan Association of use in specific influenza seasons. Available at: . [21] Brownlee GG, Fodor E. The predicted antigenicity of the haemagglutinin of the Spanish Network for the Research in Infectious Diseases (REIPI 1918 Spanish influenza pandemic suggests an avian origin. Philos Trans R Soc RD16/0016/0003); and the European Regional Development Fund Lond B Biol Sci 2001;356:1871–6. (ERDF). Cristina Andrés has been a recipient of a predoctoral [22] Xu R, Ekiert DC, Krause JC, Hai R, Crowe JE, Wilson IA. Structural basis of fellowship from the Vall d’Hebron Research Institute (PRED- preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science 2010;328:357–60. VHIR-2013). We would like to acknowledge GISAID as we used [23] Xu L, Bao L, Lv Q, Deng W, Ma Y, Li F, et al. A single-amino-acid substitution in their reference sequences. the HA protein changes the replication and pathogenicity of the 2009 pandemic A (H1N1) influenza viruses in vitro and in vivo. Virol J 2010;7:325. [24] Vazquez-Perez JA, Isa P, Kobasa D, Ormsby CE, Ramírez-Gonzalez JE, Romero- Appendix A. Supplementary material Rodríguez DP, et al. A (H1N1) pdm09 HA D222 variants associated with severity and mortality in patients during a second wave in Mexico. Virol J 2013;10:41. Supplementary data to this article can be found online at [25] Corcioli F, Arvia R, Pierucci F, Clausi V, Bonizzoli M, Peris A, et al. HA222 https://doi.org/10.1016/j.vaccine.2019.03.046. polymorphism in Influenza A(H1N1) 2009 isolates from Intensive Care Units and ambulatory patients during three influenza seasons. Virus Res 2014;180:39–42. 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