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- 2014/2015 Influenza season in an inland portuguese regionPublication . Almeida, Sofia; Rodrigues, Débora; Gouveia, Paula; Paulo, Sandra; Faria, Conceição; Pechirra, Pedro; Costa, Inês; Cristóvão, Paula; Guiomar, RaquelInfluenza surveillance is an important tool to identify emerging/reemerging strains, and defining seasonality of the circulating strains. The aim of this work is to compare the circulating strains detected by Centro Hospitalar da Cova da Beira, EPE,(CHCB) from an inland north region of Portugal, with the strains circulating among Europe during2014/2015 Influenza season. In the present review, 249 nasofaringeal swabs received in were analized by real time PCR techniques, designed to amplify Influenza A and Influenza B virus. All the positive samples were sent to the Portuguese reference Laboratory in order to subtype influenza A virus and to perform genetic and antigenic characterization of the detected viruses. During 2014/2015 season, a total of 109 positive cases (62 Influenza A and 47 Influenza B) - were detected in the studied region. In the present season, all seasonal types/subtypes of influenza virus circulated, inspite of low level circulation of the Influenza A(H1)pdm strain (~5%of the positive samples, circulating between 6/2015-9/2015 weeks). A(H3) and B strains co-circulated between weeks 1/2015 and 11/2015 with peak activity in weeks 6 and 7/2015. Influenza A, excluding A(H1)pdm, was responsible for 47% of positive cases, mainly in the last 6 weeks. Influenza B strains were responsible for 42% of positive Influenza cases, mainly in the first 7 weeks of the year. All influenza B strains characterized by the Portuguese National Laboratory were from B/Yamagata lineage. 19% of influenza B strains wereselected for genetic characterization, all were similar to B/Phuket/3073/2013 (clade 3). At the date of submission of the abstract, only 8 Influenza A strains were genetically characterized, 5 belonged to genetic subgroup 3C.2a, represented by A/Hong Kong/5738/2014, dissimilar to the current A(H3N2) vaccine strain and 3 belonged to A/Samara/73/2013 (genetic group 3C.3), antigenically similar to the current A(H3N2) vaccine The co-circulation of influenza types/subtypes was similar to the one described in Europe, but in this Portuguese region, the Influenza B circulated in first part of the epidemic period, followed by A(H3) virus at the end of epidemic period.. The genetic characterization observed in Portugal was similar to the one from ECDC data for Influenza B. Relatively to Influenza A, the majority of detected viruses were dissimilar to the 2014/2015 A(H3) vaccine virus, being a higher proportion than the observed in Europe, but more data is needed to achieve any conclusion.
- Actividade gripal em Portugal no Inverno de 2000/2001 - Análise antigénica e genética das estirpes de vírus influenzaPublication . Pechirra, Pedro; Rebelo-de-Andrade, Helena; Guiomar, Raquel; Ribeiro, Carlos; Coelho, Anabela; Pedro, Sónia; George, FranciscoAs infecções por vírus influenza são uma importante causa de morbilidade em todos os grupos etários e estão associados a uma elevada mortalidade nos idosos e nos indivíduos pertencentes a grupos de risco. No presente estudo analisaram-se os dados da vigilância epidemiológica da gripe durante o Inverno de 2000/2001. Os dados clínicos, epidemiológicos e virológicos referentes aos casos de síndroma gripal foram recolhidos através do Programa Nacional de Vigilância da Gripe que se desenvolve em colaboração com a Direcção Geral da Saúde e integra a informação obtida a partir das redes Médicos-Sentinela e Serviços de Urgência. A análise dos dados recolhidos mostram que, durante a época de Inverno de 2000/2001, a actividade gripal foi baixa, sendo o período epidémico curto e de pequena intensidade e duração. As taxas de incidência do síndrome gripal subiram acima da linha de base durante três semanas e não ultrapassaram os 74 casos por 100 000 habitantes. Os vírus influenza do tipo B foram predominantes, verificando-se a presença simultânea de vírus influenza tipo AH1 e AH3. A caracterização antigénica e genética das estirpes isoladas permitiu confirmar a semelhança entre estas estirpes virais e as estirpes vacinais e detectar a extensão dos drifts antigénicos. Saliente-se que apesar da maioria das estirpes de vírus influenza B serem idênticas antigenicamente à estirpe vacinal, a caracterização genética mostrou uma evolução dirigida para a estirpe B/ Sichuan/379/99 que viria a integrar a vacina antigripal em 2001/2002. Consequentemente, observou-se a co-circulação de duas linhagens diferentes evidenciada pela análise filogenética das estirpes B isoladas no nosso país.
- Análise antigénica e genética dos vírus da gripe: inverno 2015/2016Publication . Pechirra, Pedro; Costa, Inês; Conde, Patrícia; Cristóvão, Paula; Guiomar, RaquelA análise antigénica e genética dos vírus da gripe é um dos principais objetivos da vigilância da gripe. Na época de 2015/2016, foram caraterizadas antigénica e geneticamente, 210 e 139 estirpes virais, respetivamente, a partir de amostras biológicas recebidas através do Programa Nacional de Vigilância da Gripe e da Rede Portuguesa de Laboratórios para o Diagnóstico da Gripe. Os vírus do subtipo A(H1)pdm09, predominantes em circulação nesta época foram antigenicamente semelhantes à estirpe incluída na vacina antigripal 2015/2016. A sua caraterização genética revelou que a maioria pertence ao novo subgrupo genético 6B.1 com 4 importantes alterações na sequência peptídica da hemaglutinina em relação à estirpe vacinal. Os vírus B/Victoria, detetados no fim da epidemia de gripe, apesar de pertencerem ao grupo 1A apresentam já alguma divergência antigénica e genética em relação à estirpe B/Brisbane/60/2008 que será incluída na vacina antigripal para 2016/2017. Os vírus do subtipo A(H3), detetados em numero reduzido ao longo da época distribuíram- se pelos grupos genéticos 3C.2a e 3C.3a, com 7 e 2 substituições de aminoácidos importantes em relação à estirpe vacinal 2015/2016, respectivamente. Os vírus A(H3) caraterizados foram semelhantes antigenicamente à futura estirpe vacinal 2016/2017.
- Antigenic and genetic analysis of pandemic influenza A(H1N1) 2009 viruses from PortugalPublication . Pechirra, Pedro; Arraiolos, Ana; Conde, Patrícia; Gonçalves, Paulo; Cordeiro, Rita; Guiomar, RaquelBackround: The influenza AH1N1 2009 pandemic virus (AH1N1pdm) was first detected in Portugal in May 4th 2009. This virus had origin in a reassortment between a North American swine lineage (already a triple reassortant, circulating in pigs since the late 1990’s) and a Eurasian swine lineage. As the HA (North American swine lineage) continues to circulate in the human population, its antigenic sites will continue to be targeted by antibody-mediated selection pressure. Therefore it is important from a public health perspective, continue to characterise the HA and to monitoring the antigenic and genetic properties of the AH1N1pdm viruses in order to detect any changes and thus any necessity for selecting further vaccine candidates or changes in antiviral recommendations. In this study, is presented a genetic and antigenic characterisation of influenza AH1N1pdm viruses, isolated in Portugal over the 2009 influenza pandemic. Material and Methods: In Portugal, during the 2009 influenza pandemic, about 16500 clinical samples were tested by the National Influenza Reference Laboratory for the presence of influenza AH1N1pdm virus. From near 8000 AH1N1pdm-positive samples, 147 were isolated in MDCK-SIAT1 cell cultures and characterised antigenically by hemagglutination-inhibition assays (HI). Of these, 56 isolates were taken for sequence analysis of the HA1 gene segment. Results: Antigenically, the AH1N1pdm viruses are homogeneous, being similar to A/California/7/2009 and the later pandemic H1N1 viruses A/Bayern/69/2009 and A/Lviv/N6/2009. However, 12 of the isolated viruses show 4-fold or greater reductions in the HI titre against most of the HI sera panel. They react better with sera raised against the A/Bayern/69/2009 and A/Lviv/N6/2009. Three of these isolated viruses presented amino acid substitutions at hemagglutinin antigenic sites (G155E in epitope B; R205K in epitope D and E258D in epitope E of the HA1 subunit) and in one strain was observed the amino acid substitution V199I in the vicinity of epitope D. Changes in positions 153-157 of the HA have been highly associated with reduced HI titers with ferret antisera to the A/California/7/2009 vaccine virus. These changes usually emerge after virus propagation in cell cultures. Sequenced hemagglutinins of portuguese isolates show that these viruses, with two exceptions, belong to the clade 7, already described in the literature. As known, viruses from this clade have a S203T mutation. One of our strains, A/Lisboa/31/2009, belongs to clade 6, as presents the Q293H amino acid change. This viral strain was isolated from a patient that arrived from the USA (Boston, New York) in June 2009. Another viral strain, A/Lisboa/35/2009, belongs to an earlier clade (at least, previous to clade 4) because it don’t presents the S203T neither the Q293H in its hemagglutinin and it lacks also the V106I and N248D amino acid changes in its neuraminidase. Additionally, mutations P83S in HA present in all the portuguese isolated viruses and I321V in the majority of them have been observed in all the non-clade 1 isolates. Conclusions: The great majority of influenza AH1N1pdm viruses isolated in Portugal were similar to the vaccine strain A/California/7/2009. They were representative of the clade 7, except two strains with foreign travel history. Most of the observed amino acid changes in the HA were located at antigenic sites or in their vicinity.
- Antiviral resistance: influenza BPublication . Conde, Patrícia; Guiomar, Raquel; Cristóvão, Paula; Pechirra, PedroCurrently circulating influenza viruses are resistant to adamantanes and except for a low number of sporadic cases most are sensitive to neuraminidase inhibitors (NI). Adamantanes are ineffective against influenza B viruses and although NI-resistant influenza B viruses have been rarely reported, recently in the United States was identified one cluster of influenza B viruses with reduced susceptibility to NI and with the I221V substitution in the active site of the neuraminidase. Despite the low prevalence of oseltamivir-resistant influenza viruses, the constant evolution of influenza requires the monitoring of antiviral resistance among these viruses in the community. This is very important for the clinical management of severe influenza cases as to the detection of novel genetic markers associated with antiviral resistance. This study reports the antiviral susceptibility to neuraminidase inhibitors of influenza B viruses isolated in Portugal during the 2010/2011, 2011/2011 and 2012/2013 seasons. Over the period of 3 influenza seasons, 146 influenza B viral strains were selected for phenotypic fluorescent assays in order to assess their susceptibility to NI, oseltamivir and zanamivir. For this purpose, was determined the NI concentration required to inhibit 50% of each influenza virus neuraminidase activity (IC50). The IC50 baseline of influenza B viruses was calculated for both oseltamivir and zanamivir using the Robust Excel programme. The neuraminidase gene segments were also monitored for the presence of the main molecular markers, associated with the resistance to neuraminidase inhibitors in influenza B viruses. All analysed influenza B strains proved to be susceptible to oseltamivir and zanamivir. In the 2010/2011 season the determined IC50 values ranged from 0 to 70 nM for oseltamivir and from 0 to 11nM to zanamivir. The zanamivir IC50 median value was about 8-fold lower than oseltamivir IC50 median value. Statistical analysis revealed the presence of one outlier (B/Lisboa/13/2010, 71.08nM) for oseltamivir (2-fold reduction in susceptibility) and four minor outliers (B/Lisboa/15/2010: 9.52 nM; B/Lisboa/19/2010: 9.25 nM; B/Lisboa/51/2010: 10.94 nM and B/Lisboa/53/2010: 9.62 nM) for zanamivir (3-fold reduction in susceptibility) comparing to the median IC50 value. During the 2011/2012 season IC50 values ranged from 23.0 to 38.38 nM (oseltamivir) and from 2.97 to 9.07nM (zanamivir). This season were not found minor or major outliers. The IC50 values obtained in 2012/2013 ranged from 7.91 to 84.84 nM (oseltamivir) and from 1.48 to 5.88 nM (zanamivir). During this last season were found 6 minor and 3 major outliers for oseltamivir and 13 minor outliers for zanamivir. Along the three seasons, the median IC50 values for both NI were higher among the B/Victoria than B/Yamagata viruses. None of the actually known mutations associated with resistance of influenza B viruses to NI was found in the neuraminidase gene (R150K, D197E/N/Y, I221T/V, N294S, R374K and G407S). Influenza B viruses isolated in Portugal during the last three seasons were susceptible to the neuraminidase inhibitors. Portuguese influenza B strains revealed higher susceptibility to zanamivir than to oseltamivir, as observed in other countries. The oseltamivir IC50 values were also different between viruses from the B/Victoria and B/Yamagata lineages, however this was not statistically demonstrated due to the small number of analysed viruses. Among B/Yamagata strains the oseltamivir and zanamivir IC50 values were higher in 2011/2012 than in 2010/2011 and 2012/2013 seasons.
- Antiviral susceptibility of influenza A viruses isolated between 2009-2013, in PortugalPublication . Guiomar, Raquel; Conde, Patrícia; Cristóvão, Paula; Pechirra, PedroMonitoring the influenza antiviral susceptibility had become an important issue in the recent years. The influenza A viruses are naturally susceptible to Neuraminidase inhibitors (NAI) and M2 inhibitors (adamantanes). The increased resistance to oseltamivir and adamantanes (since 2006), the availability and clinical use of influenza antivirals enhanced the need for a close monitoring for loss of susceptibility. Since 2009, at Portuguese National influenza Centre, was developed in the scope of the National Influenza Surveillance Programme a systematic monitoring of antiviral susceptibility (oseltamivir, zanamivir and adamantine). This study shows the results of antiviral susceptibility to NAI and M2 inhibitors for influenza A viruses characterized during 4 winter seasons (2009 to 2013). The phenotypic method, NA inhibition assay, considered the “gold standard” method to determine the susceptibility to oseltamivir and zanamivir, measuring the 50% inhibitory concentrations (IC50) was performed during the study period for a selection of 119 influenza A viruses: 26 and 60 AH1pdm09 from 2009/10 and 2012/13 season, respectively, and 33 AH3 viruses from 2011/12, plus 2 AH3 from the last season were also analysed. Genotypic screening methods were performed for the most common NAI inhibition-reducing substitution, H275Y in AH1pdm09 viruses (n= 340). Matrix gene sequencing, for adamantanes susceptibility surveillance, and neuraminidase gene sequencing were performed for 64 and 119 strains, respectively. In the study period all AH1pdm09 and AH3 viruses, carried the S31N substitution in the M2 protein, which confers resistance to the adamantanes to 100% of the isolates. For the AH1pdm09 the IC50 for oseltamivir ranged from 0.45 to 2.82 (2009/2010) and from 0.15 to 3.27 (2012/2013). The resistance to oseltamivir was identified in 3 AH1pdm09 viruses that carried the H275Y substitution. Two of these viruses were isolated from patients with chronic diseases treated with oseltamivir, a pregnant woman 26 years old with a fatal outcome and an 8 years old child. One of the oseltamivir-resistant virus showed an IC5o value 250-fold higher (IC5o= 502.48) comparing to the susceptible viruses. The AH1pdm09 IC5o values for zanamivir ranged from 0.32 to 3.88 (2009/2010) and from 0.29 to 2.99 (2012/2013). The median IC50 values for oseltamivir and zanamivir were stable between 2009 and 2013. None of the AH1pdm09 isolates presented the other substitutions (D119N, I223R, N295S) associated with reduced susceptibility to NAI. The AH3 viruses analysed in 2011/2012 season showed IC5o values for oseltamivir that ranged from 0.19 to 0.90 and for zanamivir from 0.39 to 0.84. The median IC50 values for oseltamivir and zanamivir were 0.41±1.55 and 0.59±1.21 respectively. In the last season 2012/2013 only two AH3 isolates were analysed and the IC5o values were in the same range of the 2011/2012 AH3 viruses. Since 2009 all the AH3 viruses are susceptible to oseltamivir and zanamivir. None of the AH3 viruses presented the amino acid substitutions known to reduce susceptibility to NAI (E119V/I, R229K, N294S and H274Y). Influenza A viruses isolated in Portugal since 2009 are resistant to adamantanes, which are no longer indicated for influenza A treatment. Otherwise, the resistance to oseltamivir was only observed in a reduced number of strains and all the viruses show susceptibility to zanamivir. The use of conventional sequence analysis and genotypic screening methods for monitoring the molecular markers of antiviral resistance in influenza A virus provides a valuable tool for an early detection of antiviral resistant strains.
- Apresentação clínica dos casos de síndroma gripalPublication . Rodrigues, Ana Paula; Machado, Ausenda; Nunes, Baltazar; Pechirra, Pedro; Guiomar, RaquelEste trabalho pretende avaliar, com base nos dados do Programa Nacional de Vigilância de Gripe (PNVG) português, se os casos de Síndrome Gripal(SG) por influenza apresentam um quadro clínico mais grave quando comparados com outras infeções virais e com os casos negativos.
- Apresentação clínica dos casos de síndroma gripal em Portugal: gripe e outros vírus respiratóriosPublication . Rodrigues, Ana Paula; Machado, Ausenda; Nunes, Baltazar; Pechirra, Pedro; Guiomar, RaquelEste artigo pretende avaliar, com base nos dados do Programa Nacional de Vigilância de Gripe (PNVG), se os casos de síndrome gripal por influenza apresentam um quadro clínico mais grave quando comparados com os casos de síndrome gripal por outros vírus respiratórios e com os casos negativos para os vírus respiratórios em análise.
- Caraterização virológica dos vírus da gripe que circularam em Portugal na época 2014/2015Publication . Pechirra, Pedro; Costa, Inês; Cristóvão, Paula; Roque, Carla; Barreiro, Paula; Duarte, Sílvia; Machado, Ausenda; Rodrigues, Ana Paula; Nunes, Baltazar; Guiomar, RaquelIntrodução e objetivo: A monitorização contínua das propriedades antigénicas e genéticas dos vírus da gripe é essencial, quer para a seleção anual das estirpes virais a incluir na vacina, quer para identificar novas linhas de orientação da terapêutica antiviral. O presente estudo descreve as caraterísticas antigénicas e genéticas dos vírus da gripe identificados em Portugal no inverno de 2014/2015.
- Case control study for measuring influenza vaccine effectiveness in Portugal - Season 2010-11- Final reportPublication . Nunes, Baltazar; Pechirra, Pedro; Machado, Ausenda; Falcão, Isabel; Gonçalves, Paulo; Conde, Patricia; Batista, Inês; Guiomar, Raquel; Marinho Falcão, JoséEvery year, influenza virus is responsible for epidemics that affect human health causing respiratory infections that could lead to serious health complications of individuals belonging to risk groups, as well as on the functioning of health services. In order to mitigate influenza impacts, vaccination has been one of the main measures, being recognized its role in reducing the risk of developing the disease and the occurrence of their complications. Thus, since the vaccine is reformulated every season estimating the influenza vaccine effectiveness (VE) every season and in an early stage is of major importance to support public health decisions. Since 2008-2009, Portugal has been participating in I-MOVE project that aims to estimate seasonal and pandemic vaccine effectiveness during and after the influenza season. Last season, 2010-2011, Portugal once again joined the I-MOVE multi-center case control study (with the national VE study- Euroeva) together with Spain, Ireland, France, Italy, Romania, Hungary and Poland, using a common protocol and with the objective of estimate the 2010-11 seasonal influenza vaccine effectiveness respectively in the elderly (65+) and in all age groups. Additionally, using information on 2010-11 seasonal vaccine coverage in the population, it has been also proposed to estimate 2010-11 seasonal vaccine effectiveness using the screening method. Material and Methods Two different approaches were used so as to estimate vaccine effectiveness: a) For the test negative design (TND), a case-control approach was used, where laboratory confirmed influenza cases (ILI+) were compared to laboratory negative influenza ILI patients (ILI-). On a weekly basis, each GP selected systematically ILI patients (two per week from all ages and all ILI patients with 65 years and more) using the EU ILI case definition. Data on confounding factors and effect modifiers was collected using a standardized questionnaire. VE was estimated as one minus the odds ratio of being vaccinated in cases versus controls adjusted for confounders by logistic regression. b) For the screening method, the 2010-11 seasonal vaccine coverage was compared between a sample of ILI cases and a sample of ILI cases laboratory confirmed for influenza with the vaccine coverage estimated in the general population. ILI cases and ILI Positive cases were the same as the one used in the TND. Vaccine coverage in the population was obtained from a sample of 1074 households stratified by region (homogeneous allocation) selected from a dual sample frame – random digit dialing mobile and landline phones (ECOS sample). The relevant information was collected by CATI (Computer Assisted Telephone interview) with the same questionnaire – one respondent by household (proxy for the rest of the household members). VE was estimated by comparing the proportion of cases vaccinated to the vaccine coverage in the source population using the Orenstein formula and the Farrington method to adjust for confounders. For both methods an ILI patient was considered vaccinated if he/she had received one dose of the vaccine at least 14 days prior onset of symptoms. Data analysis comprised ILI positive cases selected between week 45 of 2010 and week 11 of 2011. Results In Portugal, the 2010-2011 influenza season was characterized by a mixed circulation of influenza virus. In the early beginning of the season, B type virus dominated the season, until week 1 where A(H1N1)2009 virus started to dominate. Also in circulation, but in a minor proportion was the A(H3) virus. a) Test negative design results: Considering TND results, 58 GP’s accepted to participate in the study, with 60% participation rate (35 GP’s effectively participated in the study by selecting patients). Excluding 33 ILI cases (for not meeting the inclusion criteria) the final sample for analyses consisted on 253 ILI patients with a high positive rate (57%). Among cases, 73 were positive for B virus, 69 for A(H1N1)2009 and 2 for A(H3). For analysis purposes three groups of cases were defined: All influenza, Influenza B and Influenza A(H1N1)2009 that were compared to ILI cases that tested negative for influenza virus (109 Controls). After adjustment for age group, pandemic and seasonal vaccine 2009-10 season, any chronic disease, target group, GP visits and month of onset, VE point estimates were: • 58% (CI95% -61 – 89) for All influenza • 34% (CI95% -98 – 97) for Influenza A(H1N1)2009 and • 75% (CI95% -255 – 88) for Influenza B. Nevertheless, no statistical significance was obtained for either the analysis. b) Screening method results In season 2010-2011, 903 households of the ECOS-sample were interviewed (which corresponds to 2684 individuals). The final estimated vaccine coverage was 17.5% (CI95% 15.1-20.3), with a gradient evolution since September 2010 (3.5%) till December 2010 (16.6%). Crude and adjusted VE (using the Farrington method) estimates were computed for medically attended ILI cases and ILI influenza positive cases. Overall results indicate that after adjustment for confounding (age group and presence of chronic diseases), VE point estimates decreased from 47.0% to 33.3% in ILI cases and from 70.1% to 63.7% in ILI positive cases. Due to the small sample size, no VE estimates were computed for the individuals of the vaccination target group, i.e., individuals with 65 years and more and the ones with at least one chronic disease. Conclusions Overall results obtained by the Euroeva study indicate that crude 2010-11 seasonal VE estimate against medically attended influenza was 79% (CI95% 43-94) and 70% (CI95% 32-87), respectively for the TND and screening method. After adjustment the respective VE estimates decreased: 58 (CI95% -61-89) and 64% (CI95% 17-84). These results were in accordance to the up to now published results (42-65%). The TND study was also able to provide strain specific 2010-11 seasonal VE estimates: influenza B, crude VE=87% (CI95% 41-99) and adjusted VE=75% (CI95% -98-97) and for influenza A(H1N1)2009, crude VE=74% (CI95% 14-94) and adjusted VE=34% (CI95% -254-88). These results suggest that the 2010-11 seasonal VE was lower than the monovalent A(H1N1)2009 VE estimated by the IMOVE multicenter study in the season 2009-10: 72% (CI95% 46-86). Our study was unable to estimate VE for specific seasonal vaccine target groups. This result enhances, as in previous studies, the unavoidable need for pooling data from network of VE studies with common protocol as IMOVE. Recommendations The main recommendations focused on: • To calculate sample size taking into consideration: o the context of the multicentre case-control study: minimum sample size per site in order to assure a minimum homogeneity for pooled analysis; o different expected VE point estimates, i.e. for low, medium and high VE; o minimum set of factors for stratified analysis; o the adjustment for confounders. • To increase sample size, mainly in the elderly population (aged 65 years or more); • To increase the total number of participating GP’s in the study by exploring other sources of GP’s recruitment; • To study the inclusion of the population based vaccine coverage uncertainty in the screening method; • To explore with participating GP’s the best way to obtain estimates of the Euroeva ILI sample fraction. Finally we also recommend continuing the harmonization of the study designs between participating countries with the multi-centre study objective.