Browsing by Author "Conde, Patrícia"
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- 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.
- Case control study for measuring influenza vaccine effectiveness in Portugal - Final report Season 2009-10Publication . Nunes, Baltazar; Guiomar, Raquel; Machado, Ausenda; Falcão, Isabel; Gonçalves, Paulo; Conde, Patrícia; Batista, Inês; Dias, Carlos Matias; Marinho Falcão, JoséO Departamento de Epidemiologia em conjunto com o Departamento de Doenças Infecciosas do INSA realizaram um estudo, na época de 2009-2010, que teve por objectivo estimar a efectividade da vacina sazonal (em indivíduos com 65 e mais anos) e pandémica (todas as idades). O estudo contou com a participação de: Portugal, Espanha, Irlanda, França, Itália, Hungria e Roménia. Este estudo surge enquadrado no projecto europeu multicêntrico - I-MOVE- Monitoring influenza vaccine efectiveness during influenza seasons and pandemics in the European Union, coordenado pela EpiConcept SARL e financiado pelo ECDC, que pretende estimar a efectividade da vacina sazonal e pandémica durante e após a época de gripe.
- Characterisation of influenza B viruses circulating in Portugal during the 2010/2011 influenza seasonPublication . Pechirra, Pedro; Conde, Patrícia; Romero, Carla; Ribeiro, Carlos; Gonçalves, Paulo; Guiomar, Raquel
- Characterisation of respiratory disease during the 2010/2011 influenza winter season in Portugal: Contribute of the Laboratory Network for the Diagnosis of Influenza A(H1N1)2009 InfectionPublication . Pechirra, Pedro; Gonçalves, Paulo; Conde, Patrícia; Guiomar, Raquel; Duque, Vítor; Vaz, João; Ribeiro, Graça; Cabral, Rita; Mota Vieira, Luísa; Almeida Santos, Madalena; Silvestre, Maria José; Pimentel Couto, Ana Rita; Bruges Armas, Jacome; Castro, Ana Paula; Ramos, Maria Helena; Janeiro, André; Mimoso, Paula; Marcelino, Rute; Fernandes, Aida; Milho, Luís; Rego, João; Beleza, Álvaro; Barreto, Maria do Rosário; Carvalho, Dinah; Ribeiro, Carlos Manuel; Fernandes, Paula; Andrade, Graça; Sobrinho Simões, Joana; Costa, Maria do Rosário; Guimarães, João Tiago; Corte Real, Rita; Branquinho, Paula; Caldeira, Filomena; Maurílio, Manuel
- Cross-protection to new drifted influenza A(H3) viruses and prevalence of protective antibodies to seasonal influenza, during 2014 in PortugalPublication . Guiomar, Raquel; Pereira da Silva, Susana; Conde, Patrícia; Cristóvão, Paula; Maia, Ana Carina; Pechirra, Pedro; Rodrigues, Ana Paula; Nunes, Baltazar; Milho, Luís; Coelho, Ana Paula; Fernandes, Aida; Caseiro, Paula; Rodrigues, Fernando; Correia, Lurdes; Pereira-Vaz, João; Almeida, Sofia; Branquinho, Paula; Côrte-Real, Rita; Viseu, Regina; Peres, Maria João; Sanches, Raquel; Dantas, Filipa; Freitas, Ludovina; Andrade, Graça; Maurílio, Manuel; Caldeira, Filomena; Cabral Veloso, Rita; Mota-Vieira, Luísa; Soares, Marta; Couto, Ana Rita; Bruges-Armas, Jácome; Mouro Pinto, Rita; Sobrinho Simões, Joana; Rosário Costa, Maria; Guimarães, João Tiago; Martins, Luís; Cunha, MárioIntroduction: 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 crossprotection 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 65 years 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.
- Cross-protection to new drifted influenza A(H3) viruses and prevalence of protective antibodies to seasonal influenza, during 2014 in PortugalPublication . Guiomar, Raquel; Pereira da Silva, Susana; Conde, Patrícia; Cristóvão, Paula; Maia, Ana Carina; Pechirra, Pedro; Rodrigues, Ana Paula; Nunes, Baltazar; Milho, Luís; Coelho, Ana Paula; Fernandes, Aida; Caseiro, Paula; Rodrigues, Fernando; Correia, Lurdes; Pereira-Vaz, João; Almeida, Sofia; Branquinho, Paula; Côrte-Real, Rita; Viseu, Regina; Peres, Maria João; Sanches, Raquel; Dantas, Filipa; Freitas, Ludovina; Andrade, Graça; Maurílio, Manuel; Caldeira, Filomena; Cabral Veloso, Rita; Mota-Vieira, Luísa; Soares, Marta; Couto, Ana Rita; Bruges-Armas, Jácome; Mouro Pinto, Rita; Sobrinho Simões, Joana; Costa, Maria do Rosário; Guimarães, João Tiago; Martins, Luís; Cunha, MárioIntroduction: 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 65 years 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.
- Diagnóstico diferencial de vírus respiratórios em casos de síndroma gripal no inverno 2013/2014Publication . Pechirra, Pedro; Cristóvão, Paula; Maia, Ana Carina; Conde, Patrícia; Furtado, Cristina; Guiomar, Raquel