Epidemiology and genetic variability of respiratory syncytial virus in Portugal, 2014-2018.

INTRODUCTION: Respiratory syncytial virus (RSV) is associated with substantial morbidity and mortality since it is a predominant viral agent causing respiratory tract infections in infants, young children and the elderly. Considering the availability of the RSV vaccines in the coming years, molecular understanding in RSV is necessary. OBJECTIVE: The objective of the present study was to describe RSV epidemiology and genotype variability in Portugal during the 2014/15-2017/18 period. MATERIAL AND METHODS: Epidemiological data and RSV-positive samples from patients with a respiratory infection were collected through the non-sentinel and sentinel influenza surveillance system (ISS). RSV detection, subtyping in A and B, and sequencing of the second hypervariable region (HVR2) of G gene were performed by molecular methods. Phylogenetic trees were generated using the Neighbor-Joining method and p-distance model on MEGA 7.0. RESULTS: RSV prevalence varied between the sentinel (2.5%, 97/3891) and the non-sentinel ISS (20.7%, 3138/16779), being higher (P < 0.0001) among children aged <5 years. Bronchiolitis (62.9%, 183/291) and influenza-like illness (24.6%, 14/57) were associated (P < 0.0001) with RSV laboratory confirmation among children aged <6 months and adults ≥65 years, respectively. The HVR2 was sequenced for 562 samples. RSV-A (46.4%, 261/562) and RSV-B (53.6%, 301/562) strains clustered mainly to ON1 (89.2%, 233/261) and BA9 (92%, 277/301) genotypes, respectively, although NA1 and BA10 were also present until 2015/2016. CONCLUSION: The sequence and phylogenetic analysis reflected the relatively high diversity of Portuguese RSV strains. BA9 and ON1 genotypes, which have been circulating in Portugal since 2010/2011 and 2011/2012 respectively, predominated during the whole study period.

Performance of surveillance case definitions for respiratory syncytial virus infections through the sentinel influenza surveillance system, Portugal, 2010 to 2018.

BackgroundWell-established influenza surveillance systems (ISS) can be used for respiratory syncytial virus (RSV) surveillance. In Portugal, RSV cases are detected through the ISS using the European Union (EU) influenza-like illness (ILI) case definition.AimTo investigate clinical predictors for RSV infection and how three case definitions (EU ILI, a modified EU acute respiratory infection, and one respiratory symptom) performed in detecting RSV infections in Portugal.MethodsThis observational retrospective study used epidemiological and laboratory surveillance data (October 2010-May 2018). Associations between clinical characteristics and RSV detection were analysed using logistic regression. Accuracy of case definitions was assessed through sensitivity, specificity, and area under the receiver operating characteristic curve (AUC). A 0.05 significance level was accepted.ResultsThe study involved 6,523 persons, including 190 (2.9%) RSV cases. Among 183 cases with age information, RSV infection was significantly more frequent among individuals < 5 years (n = 23; 12.6%) and ≥ 65 years (n = 45; 24.6%) compared with other age groups (p < 0.0001). Cough (odds ratio (OR): 2.4; 95% confidence interval (CI): 1.2-6.5) was the best RSV-infection predictor considering all age groups, while shortness of breath was particularly associated with RSV-positivity among ≤ 14 year olds (OR: 6.7; 95% CI: 2.6-17.4 for 0-4 year olds and OR: 6.7; 95% CI: 1.5-28.8 for 5-14 year olds). Systemic symptoms were significantly associated with RSV-negative and influenza-positive cases. None of the case definitions were suitable to detect RSV infections (AUC = 0.51).ConclusionTo avoid underestimating the RSV disease burden, RSV surveillance within the Portuguese sentinel ISS would require a more sensitive case definition than ILI and, even a different case definition according to age.

INSaFLU: an automated open web-based bioinformatics suite "from-reads" for influenza whole-genome-sequencing-based surveillance.

BACKGROUND: A new era of flu surveillance has already started based on the genetic characterization and exploration of influenza virus evolution at whole-genome scale. Although this has been prioritized by national and international health authorities, the demanded technological transition to whole-genome sequencing (WGS)-based flu surveillance has been particularly delayed by the lack of bioinformatics infrastructures and/or expertise to deal with primary next-generation sequencing (NGS) data. RESULTS: We developed and implemented INSaFLU ("INSide the FLU"), which is the first influenza-oriented bioinformatics free web-based suite that deals with primary NGS data (reads) towards the automatic generation of the output data that are actually the core first-line "genetic requests" for effective and timely influenza laboratory surveillance (e.g., type and sub-type, gene and whole-genome consensus sequences, variants' annotation, alignments and phylogenetic trees). By handling NGS data collected from any amplicon-based schema, the implemented pipeline enables any laboratory to perform multi-step software intensive analyses in a user-friendly manner without previous advanced training in bioinformatics. INSaFLU gives access to user-restricted sample databases and projects management, being a transparent and flexible tool specifically designed to automatically update project outputs as more samples are uploaded. Data integration is thus cumulative and scalable, fitting the need for a continuous epidemiological surveillance during the flu epidemics. Multiple outputs are provided in nomenclature-stable and standardized formats that can be explored in situ or through multiple compatible downstream applications for fine-tuned data analysis. This platform additionally flags samples as "putative mixed infections" if the population admixture enrolls influenza viruses with clearly distinct genetic backgrounds, and enriches the traditional "consensus-based" influenza genetic characterization with relevant data on influenza sub-population diversification through a depth analysis of intra-patient minor variants. This dual approach is expected to strengthen our ability not only to detect the emergence of antigenic and drug resistance variants but also to decode alternative pathways of influenza evolution and to unveil intricate routes of transmission. CONCLUSIONS: In summary, INSaFLU supplies public health laboratories and influenza researchers with an open "one size fits all" framework, potentiating the operationalization of a harmonized multi-country WGS-based surveillance for influenza virus. INSaFLU can be accessed through https://insaflu.insa.pt .

Respiratory infections in elderly people: Viral role in a resident population of elderly care centers in Lisbon, winter 2013-2014.

OBJECTIVE: The aim of this study was to analyze the etiology and clinical consequences of viral respiratory infections in 18 elderly care centers (ECC) in Lisbon, which housed a total of 1022 residents. METHODS: Nasopharyngeal swabs were collected whenever an elderly had symptoms of acute respiratory infections (ARI). PCR and RT-PCR were performed for influenza A/B, human parainfluenza virus 1-4, adenovirus, human metapneumovirus (HMPV), respiratory syncytial virus (RSV), rhinovirus, enterovirus, human coronavirus and human Bocavirus (HBoV). Array cards for atypical bacteria were also used in severe cases. RESULTS: In total, 188 episodes of ARI were reported, being rhinovirus the most frequently detected (n=53), followed by influenza A(H3) (n=19) and HBoV (n=14). Severe infections were reported in 19 patients, 11 of which were fatal, Legionela pneumophila, rhinovirus, HMPV and RSV associated with these fatalities. Nine influenza strains were analyzed, all antigenically dissimilar from vaccine strain 2013/14. "Age", "HMPV" and "Respiratory disease" showed an association with severe infection. CONCLUSIONS: In this study an etiologic agent could be found in 60% of the acute respiratory episodes. These data provides information about the circulating viruses in ECC and highlights the importance of searching both viruses and atypical bacteria in severe ARI.

Cross-protection to new drifted influenza A(H3) viruses and prevalence of protective antibodies to seasonal influenza, during 2014 in Portugal.

INTRODUCTION: Immune profile for influenza viruses is highly changeable over time. Serological studies can assess the prevalence of influenza, estimate the risk of infection, highlight asymptomatic infection rate and can also provide data on vaccine coverage. The aims of the study were to evaluate pre-existing cross-protection against influenza A(H3) drift viruses and to assess influenza immunity in the Portuguese population. MATERIALS AND METHODS: We developed a cross-sectional study based on a convenience sample of 626 sera collected during June 2014, covering all age groups, both gender and all administrative health regions of Portugal. Sera antibody titers for seasonal and new A(H3) drift influenza virus were evaluated by hemagglutination inhibition assay (HI). Seroprevalence to each seasonal influenza vaccine strain virus and to the new A(H3) drift circulating strain was estimated by age group, gender and region and compared with seasonal influenza-like illness (ILI) incidence rates before and after the study period. RESULTS: Our findings suggest that seroprevalences of influenza A(H3) (39.9%; 95% CI: 36.2-43.8) and A(H1)pdm09 (29.7%; 95% CI: 26.3-33.4) antibodies were higher than for influenza B, in line with high ILI incidence rates for A(H3) followed by A(H1)pdm09, during 2013/2014 season. Low pre-existing cross-protection against new A(H3) drift viruses were observed in A(H3) seropositive individuals (46%). Both against influenza A(H1)pdm09 and A(H3) seroprotection was highest in younger than 14-years old. Protective antibodies against influenza B were highest in those older than 65years old, especially for B/Yamagata lineage, 33.3% (95% CI: 25.7-41.9). Women showed a high seroprevalence to influenza, although without statistical significance, when compared to men. A significant decreasing trend in seroprotection from north to south regions of Portugal mainland was observed. CONCLUSIONS: Our results emphasize that low seroprotection increases the risk of influenza infection in the following winter season. Seroepidemiological studies can inform policy makers on the need for vaccination and additional preventive measures.

Hospitalization Risk Due to Respiratory Illness Associated with Genetic Variation at IFITM3 in Patients with Influenza A(H1N1)pdm09 Infection: A Case-Control Study.

BACKGROUND: Recent studies suggest an association between the Interferon Inducible Transmembrane 3 (IFITM3) rs12252 variant and the course of influenza infection. However, it is not clear whether the reported association relates to influenza infection severity. The aim of this study was to estimate the hospitalization risk associated with this variant in Influenza Like Illness (ILI) patients during the H1N1 pandemic influenza. METHODS: A case-control genetic association study was performed, using nasopharyngeal/oropharyngeal swabs collected during the H1N1 pandemic influenza. Laboratory diagnosis of influenza infection was performed by RT-PCR, the IFITM3 rs12252 was genotyped by RFLP and tested for association with hospitalization. Conditional logistic regression was performed to calculate the confounder-adjusted odds ratio of hospitalization associated with IFITM3 rs12252. RESULTS: We selected 312 ILI cases and 624 matched non-hospitalized controls. Within ILI Influenza A(H1N1)pdm09 positive patients, no statistical significant association was found between the variant and the hospitalization risk (Adjusted OR: 0.73 (95%CI: 0.33-1.50)). Regarding ILI Influenza A(H1N1)pdm09 negative patients, CT/CC genotype carriers had a higher risk of being hospitalized than patients with TT genotype (Adjusted OR: 2.54 (95%CI: 1.54-4.19)). CONCLUSIONS: The IFITM3 rs12252 variant was associated with respiratory infection hospitalization but not specifically in patients infected with Influenza A(H1N1)pdm09.

Vaccine effectiveness in preventing laboratory-confirmed influenza in primary care patients in a season of co-circulation of influenza A(H1N1)pdm09, B and drifted A(H3N2), I-MOVE Multicentre Case-Control Study, Europe 2014/15.

Influenza A(H3N2), A(H1N1)pdm09 and B viruses co-circulated in Europe in 2014/15. We undertook a multicentre case-control study in eight European countries to measure 2014/15 influenza vaccine effectiveness (VE) against medically-attended influenza-like illness (ILI) laboratory-confirmed as influenza. General practitioners swabbed all or a systematic sample of ILI patients. We compared the odds of vaccination of ILI influenza positive patients to negative patients. We calculated adjusted VE by influenza type/subtype, and age group. Among 6,579 ILI patients included, 1,828 were A(H3N2), 539 A(H1N1)pdm09 and 1,038 B. VE against A(H3N2) was 14.4% (95% confidence interval (CI): -6.3 to 31.0) overall, 20.7% (95%CI: -22.3 to 48.5), 10.9% (95%CI -30.8 to 39.3) and 15.8% (95% CI: -20.2 to 41.0) among those aged 0-14, 15-59 and  ≥60 years, respectively. VE against A(H1N1)pdm09 was 54.2% (95%CI: 31.2 to 69.6) overall, 73.1% (95%CI: 39.6 to 88.1), 59.7% (95%CI: 10.9 to 81.8), and 22.4% (95%CI: -44.4 to 58.4) among those aged 0-14, 15-59 and  ≥60 years respectively. VE against B was 48.0% (95%CI: 28.9 to 61.9) overall, 62.1% (95%CI: 14.9 to 83.1), 41.4% (95%CI: 6.2 to 63.4) and 50.4% (95%CI: 14.6 to 71.2) among those aged 0-14, 15-59 and ≥60 years respectively. VE against A(H1N1)pdm09 and B was moderate. The low VE against A(H3N2) is consistent with the reported mismatch between circulating and vaccine strains.

Estimates of 2012/13 influenza vaccine effectiveness using the case test-negative control design with different influenza negative control groups.

BACKGROUND: In recent years several reports of influenza vaccine effectiveness (VE) have been made early for public health decision. The majority of these studies use the case test-negative control design (TND), which has been showed to provide, under certain conditions, unbiased estimates of influenza VE. Nevertheless, discussions have been taken on the best influenza negative control group to use. The present study aims to contribute to the knowledge on this field by comparing influenza VE estimates using three test-negative controls: all influenza negative, non-influenza respiratory virus and pan-negative. METHODS: Incident ILI patients were prospectively selected and swabbed by a sample of general practitioners. Cases were ILI patients tested positive for influenza and controls ILI patients tested negative for influenza. The influenza negative control group was divided into non-influenza virus control group and pan-negative control group. Data were collected on vaccination status and confounding factors. Influenza VE was estimated as one minus the odds ratio of been vaccinated in cases versus controls adjusted for confounding effect by logistic regression. RESULTS: Confounder adjusted influenza VE against medically attended laboratory-confirmed influenza was 68.4% (95% CI: 20.7-87.4%) using all influenza negatives controls, 82.1% (95% CI: 47.6-93.9%) using non-influenza controls and 49.4% (95% CI: -44.7% to 82.3%) using pan-negative controls. CONCLUSIONS: Influenza VE estimates differed according to the influenza negative control group used. These results are in accordance with the expected under the hypothesis of differential viral interference between influenza vaccinated and unvaccinated individuals. Given the wide importance of TND study further studies should be conducted in order to clarify the observed differences.

[National Influenza Surveillance Programme: results of influenza activity in Portugal in the 2010/2011 season]. / Programa Nacional de Vigilância da Gripe: resultados da atividade gripal em Portugal na época 2010/2011.

INTRODUCTION: Epidemiological surveillance of influenza, a disease associated with high mortality in the elderly and individuals' belonging to risk groups, is essential for the characterization of influenza epidemics as well as for the monitoring of outbreaks and the emergence of viral strains resistant to antivirals. MATERIAL AND METHODS: In the present study was analyzed the influenza surveillance data from 2010/2011 winter. The clinical, epidemiological and virological data related to cases of flu-like syndrome have been collected through the National Influenza Surveillance Programme, coordinated by the National Influenza Reference Laboratory (LNRVG) in collaboration with the Department of Epidemiology(DEP) of the National Institute of Health Doutor Ricardo Jorge (INSA) and the Directorate-General for Health (DGS). RESULTS: From the analysis of the data collected during the 2010/2011 winter season, influenza activity was moderate / high with an epidemic period of 8 weeks between week 50/2010 and 5 / 2011, with a peak of 121, 12 cases per 100 000 population in week 52/2010. DISCUSSION: The influenza B viruses (Victoria lineage) predominated in the early period to week 1 / 2011, when became to predominate influenza A(H1)pdm09 viruses. The largest proportion of cases of influenza was found in the age group of children between 5 and 14 years old. The viruses characterized antigenically and genetically were similar to strains included in the seasonal influenza vaccine 2010/2011, presenting some amino acid substitutions in antigenic sites. Most strains of A(H1)pdm09 influenza virus still remain sensitive to oseltamivir and zanamivir, although were detected sporadic cases of oseltamivir resistant viruses. To date, the LNRVG detected the presence of the H275Y substitution in the neuraminidase gene, associated with oseltamivir resistance, in three virus A(H1)pdm09.For one of them, oseltamivir resistance was confirmed by phenotypic assays. CONCLUSION: Despite fears associated with the emergence of a new pandemic virus (rapid spread, high morbidity and mortality), the clinical and epidemiological characteristics of infection with influenza A(H1)pdm09, proved in the course of two seasons (2009/2010 and 2010/2011) to be very similar to seasonal flu. Is however noteworthy that during the pandemic season(2009/2010) the influenza virus A (H1N1) pdm09 were linked with particular characteristics with regard to their temporal distribution, age groups most affected,severe infections and death and associated risk factors.

I-MOVE multi-centre case control study 2010-11: overall and stratified estimates of influenza vaccine effectiveness in Europe.

BACKGROUND: In the third season of I-MOVE (Influenza Monitoring Vaccine Effectiveness in Europe), we undertook a multicentre case-control study based on sentinel practitioner surveillance networks in eight European Union (EU) member states to estimate 2010/11 influenza vaccine effectiveness (VE) against medically-attended influenza-like illness (ILI) laboratory-confirmed as influenza. METHODS: Using systematic sampling, practitioners swabbed ILI/ARI patients within seven days of symptom onset. We compared influenza-positive to influenza laboratory-negative patients among those meeting the EU ILI case definition. A valid vaccination corresponded to > 14 days between receiving a dose of vaccine and symptom onset. We used multiple imputation with chained equations to estimate missing values. Using logistic regression with study as fixed effect we calculated influenza VE adjusting for potential confounders. We estimated influenza VE overall, by influenza type, age group and among the target group for vaccination. RESULTS: We included 2019 cases and 2391 controls in the analysis. Adjusted VE was 52% (95% CI 30-67) overall (N = 4410), 55% (95% CI 29-72) against A(H1N1) and 50% (95% CI 14-71) against influenza B. Adjusted VE against all influenza subtypes was 66% (95% CI 15-86), 41% (95% CI -3-66) and 60% (95% CI 17-81) among those aged 0-14, 15-59 and ≥60 respectively. Among target groups for vaccination (N = 1004), VE was 56% (95% CI 34-71) overall, 59% (95% CI 32-75) against A(H1N1) and 63% (95% CI 31-81) against influenza B. CONCLUSIONS: Results suggest moderate protection from 2010-11 trivalent influenza vaccines against medically-attended ILI laboratory-confirmed as influenza across Europe. Adjusted and stratified influenza VE estimates are possible with the large sample size of this multi-centre case-control. I-MOVE shows how a network can provide precise summary VE measures across Europe.

Heterogeneous selective pressure acting on influenza B Victoria- and Yamagata-like hemagglutinins.

As a consequence of immune pressure, influenza virus hemagglutinin presents some of its amino acids under positive selection. Several authors have reported the existence of influenza A hemagglutinin codons under positive selective pressure (PSP). In this framework, the present work objectives were to demonstrate the presence of PSP and evaluate its effects on Victoria- and Yamagata-like influenza B viruses. Methodology adopted consisted in estimating the acceptance rate of nonsynonymous substitutions (omega = dN/dS) that describe the strength of selective pressure and identifying codons that may be positively selected, applying a set of continuous-time Markov chain codon-substitution models. Two groups of HA1 sequences (140 from Yamagata and 60 from Victoria lineage) were used. All the model maximum-likelihood estimates were obtained using codeml software application (PAML 3.15). The hypothesis of no existence of sites under PSP was rejected for both lineages (p < 0.001), using likelihood ratio tests. These results demonstrate the presence of positive selection acting on hemagglutinin of both Yamagata- and Victoria-like influenza B viruses. Several different sites were identified to be under PSP on Yamagata and Victoria hemagglutinins. Sites found with a posterior probability > 0.95 were codons 197 and 199 in both lineages, codon 75 in the Yamagata lineage, and codon 129 in the Victoria lineage. The detected amino acids are located at or near antigenic sites in influenza A virus H3 hemagglutinin.

Characterization of influenza A/Fujian/411/2002(H3N2)-like viruses isolated in Portugal between 2003 and 2005.

In Portugal, influenza surveillance is achieved through the National Influenza Surveillance Programme (NISP), in close collaboration with other European and global surveillance networks. The NISP integrates epidemiological, clinical and virological data based on the information collected by a Network of Sentinel Medical Practitioners and by a network of Emergency Units of Hospitals and Health Care Centres. In this study, genetic and antigenic characterization of influenza A viruses of the A/Fujian/411/2002 lineage, isolated during the 2003/2004 and 2004/2005 influenza winter seasons, in the context of the NISP, are described. Antigenic analysis of A/Fujian/411/2002-like viruses, first detected and isolated during the 2003/2004 winter season, revealed a close similarity with the reference strains A/Kumamoto/102/2002 and A/Wyoming/3/2003. Genetic analysis confirmed this similarity and revealed two different phylogenetic branches. The 2004/2005 influenza A(H3) isolates formed, both antigenic and genetically, a more homogeneous group and were closely related to A/Oslo/807/2004 and A/California/7/2004. During this season, the characterization of the influenza viral strains has shown continuous evolution to variants close related to A/Oslo/807/2004. The majority of amino acid substitutions detected in the haemagglutinin occurred at antigenic sites. This study reflects the contribution of individual countries for the surveillance and knowledge of the molecular epidemiology of the infection, essential for a concerted action towards the global monitoring of the disease. It also reflects the importance of constant monitoring of genetic and antigenic characteristics of circulating influenza strains, which will certainly be a major contribution to the formulation of influenza vaccines.

Molecular characterization of the HA gene of influenza type B viruses.

Nucleotide sequences of the HA1 subunit of influenza B viruses isolated in Portugal between 1994 and 2003 influenza winter seasons were analyzed by the Neighbor-Joining algorithm and rates of HA1 evolution estimated by linear regression. From 1994 to 2002, all influenza B viruses studied were of the Yamagata lineage. Strains isolated from 1994 to 1996, 1996 to 1999, and 1999 to 2002 revealed a high similarity with B/Beijing/184/93, B/Yamanashi/166/98, and B/Sichuan/379/99, respectively, and strains isolated during 1994-1995, 1996-1997, and 1998-1999 clustered in more than one branch of the phylogenetic tree. Victoria-related strains reappeared during 2002/2003 and formed only one branch in the phylogenetic tree revealing a closer relationship to B/Shandong/7/97. Evolutionary rates for strains from the Yamagata lineage were estimated as 3.82x10(-3) nucleotides/site/year and 2.62x10(-3) nucleotides/site/year for Victoria-related strains. In order to identify putative influenza B HA1 codons under selective pressure, a codon-substitution model for heterogeneous selective pressure at amino acid sites was used. A percentage of 97.3% of codons under negative selective pressure and 2.7% of codons under positive selective pressure (omega=dN/dS=2.65) were estimated, with posterior probability higher than 0.90. Amino acid sites 75, 197, and 199 were found more likely to be under positive selective pressure.

[Influenza activity 2000/2001]. / Actividade gripal em Portugal no inverno de 2000/2001: análise antigénica e genética das estirpes de vírus influenza.

Influenza viral infections are an important cause of morbidity in all age groups and are associated with a high mortality rate amongst the elderly and in risk groups. The current study analyses data from the epidemiological surveillance of influenza during the 2000/2001-winter season. Clinical, epidemiological and virological data relative to cases of influenza syndrome were collected via the National Influenza Surveillance Scheme, in collaboration with the General Directory of Health and integrates the information obtained by the Network of Sentinel Medical Practitioners and Emergency Units. The data analysis hereby obtained shows a low influenza activity during the 2000/2001-winter season, with an epidemiological period characterized by small duration and intensity. The incidence rate of influenza-like illnesses increased above the base line during three weeks and didn't surpass 74 cases per 1,000,000 inhabitants. Influenza B viruses were predominant with the simultaneous presence of influenza A (H1 and H3). The antigenic and genetic characterisation of the isolates allowed the confirmation of the similarity between these viral strains and the vaccine strains as well as the extent of the antigenic drift. Despite the antigenic similarity between the majority of influenza B isolates and the vaccine strains it's noticeable that the genetic characteristics showed an evolution directed towards the B/Sichuan/379/99 strain, which would later be incorporated in the 2001/2002 influenza vaccine. The co-circulation of two different lineages was consequently observed by the phylogenetic analysis of the B strains isolated in our country.