DDI - Posters/abstracts em congressos internacionais
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- Analysis of longitudinal nef sequence variation throughout HIV-2 infectionPublication . Remédios, M.; Paixão, E.; Feliciano, H.; Silva-Graça, A.; Pádua, E.Background: Human Immunodeficiency Virus type 1 (HIV-1) and type 2 (HIV-2) may case a severe immunodeficiency in humans (AIDS). However HIV-2 is more frequently associated with lower levels of transmission and disease progression, compared with HIV-1 infections. The role of nef gene in vivo during the development of AIDS has been clearly demonstrated in simian immunodeficiency virus infected Rhesus macaques model, but the determinants which play a role in the pathogenesis of HIV are relatively poorly understood. However, even less is known about the role of nef in HIV-2 infections. Methods: In this study, it was analyzed the variation of 48 nef gene sequences, obtained from samples taken between 1994 and 2009, corresponding to 17 HIV-2-infected individuals with different clinical stages of infection. The sequences obtained by Nested PCR were classified by phylogenetic analysis and the functional protein motifs, described as important in CD4 and MHC-I downregulation and in viral infectivity were also analyzed. Results: In all individuals were identified nef sequences from group A of HIV-2, which encoded possible functional and complete protein. There was a greater conservation of residues in the Nef sequences of individuals in the symptomatic stage (63%), comparatively to individuals in the asymptomatic stage (19%). While some functional motifs (MGxxxS1, DDDD93, RR137 and DD205) and also residues (G128, I141 and L142) remained conserved, others (YSRF39, LRAR21, PxxP101, EE185) revealed changes during the follow-up period. The PxxP motif exhibited wide inter-individual variation in vivo from an HIV-1-like tetra-proline motif (PxxP)3 to disruption of the minimal core PxxPLR motif. The disruption was observed in 11 sequences exclusively from asymptomatic individuals (p=0.021). The sequence motif variation towards tetra-proline configuration was observed in 2 symptomatic individuals during time of infection. The results also revealed the existence of a negative selective pressure, as well as codons under positive pressure in the sequences. Conclusion: In this HIV-2-infected individuals studied, it was observed a need for a greater degree of Nef protein conservation in a symptomatic phase. Sequences altered and potentially critical for the Nef function in vivo, in earlier stages of infection, may contribute at some level to a different pattern in viral pathogenesis and disease progression.
- Seasonal and pandemic patterns of Influenza in PortugalPublication . Gonçalves, Paulo; Nunes, Baltazar; Paixão, Eleonora; Cordeiro, Rita; Pechirra, Pedro; Conde, Patrícia; Arraiolos, Ana; Furtado, Cristina; Guiomar, RaquelThe National Influenza Reference Laboratory has been collecting data on influenza activity in Portugal since 1957 through the National Influenza Surveillance Programme, including information on clinical and virological characteristics of the disease, allowing the estimation of weekly incidence rates for influenza-like illness (ILI). This information has not only been used by the National Health Authorities for the management of the disease, in its several aspects, but has also been contributing to the study of influenza by the World Health Organisation. Particularly during the past decade, the world had been preparing for a long awaited influenza pandemic, which characteristics could not been foreseen but was feared to have potentially devastating consequences. In April 2009 a new strain of influenza A(H1N1) virus of swine origin disseminated throughout the world, resulting in the first pandemic of the XXI century. To face the increasing number of diagnosis being requested, a Network of Associated Laboratories dedicated exclusively to the diagnosis of the new influenza A(H1N1) pandemic virus was activated in our country. The data on influenza collected over the past two influenza seasons, through the National Influenza Surveillance Programme and the Network of Associated Laboratories, is presented and compared. 2. Materials and methods Over 3000 ILI cases were notified to the National Influenza Reference Laboratory and to the Department of Epidemiology of the National Institute of Health, in the context of the National Influenza Surveillance Programme, from week 38/2008 through week 20/2010. The distribution by age group, gender and region, and the signs and/or symptoms present were analysed. Nasopharyngeal swabs were collected for virological characterisation of influenza viruses circulating during this period. The intensity and duration of the epidemic periods were described based on the weekly incidence rates for ILI and as a function of a defined baseline. From week 17/2009 through week 20/2010, over 53.800 ILI cases and respective biological specimens were also notified and analysed by the Network of Associated Laboratories. 3. Results Seasonal AH1, AH3 and B influenza viruses circulating in Portugal during the 2008/2009 season were replaced by the new influenza A(H1N1) pandemic virus since the beginning of the pandemic in the country on week 17/2009. When considering the data obtained though the National Influenza Surveillance Programme, lower ILI incidence rates, lower percentage of confirmed influenza cases (particularly in the population over 15 years) and fewer symptoms presented at the time of notification were observed during the 2009/2010 pandemic. The large volume of cases analysed through the Network of Associated Laboratories is currently under evaluation and will be presented at a later stage. 4. Conclusions Facing the circulation of a new virus and the threat that this could impose to the population and to the health care system, the total number of ILI cases reported and analysed in our country during the 2009/2010 winter boosted to numbers not seen in previous influenza seasons. It is a fact that the 2009/2010 pandemic has had a significant impact in Portugal in many areas, such as the adoption of health-care regulations, availability of health-care facilities, vaccination strategies, and public action/awareness. However, in terms of pattern of disease, the data collected through the National Influenza Surveillance Programme may reveal a different reality. The data analysed suggests that the 2009/2010 pandemic was milder than the previous influenza seasons. Preliminary analysis of the data collected through the Network of Associated Laboratories appears to corroborate these findings.
- Sequence analysis of resistance markers in pandemic influenza A (H1N1) 2009 viruses isolated in PortugalPublication . Pechirra, Pedro; Arraiolos, Ana; Conde, Patrícia; Gonçalves, Paulo; Cordeiro, Rita; Guiomar, RaquelBackround: In April 2009, a new influenza A (H1N1) virus (AH1N1pdm) was detected circulating in humans. Its rapid widespread transmission led to the declaration by the WHO of an influenza pandemic. 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 (which contributed to the AH1N1pdm with the neuraminidase (NA) and matrix (MP) gene segments). The NA inhibition assay is the base test to determine the susceptibility to NA inhibitors (NAI), however it requires virus isolation, and the consequent detection of a resistant viral strain requires confirmation by sequencing of the NA gene segment present in the original biological product. 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, 153 were isolated in MDCK cells and of these 53 isolates were taken for sequence analysis of the NA and MP gene segments. The NA inhibition assays were performed by the WHO collaborating centre (London). Results: As all AH1N1pdm viruses, sequenced Portuguese isolates carried the S31N change in the M2 protein, which confers resistance to the adamantanes. The most common mutation in N1 neuraminidase associated with resistance to oseltamivir is the H275Y change. From our 53 sequenced isolates, only one carried this mutation. This was from an 8-years-old child with chronic diseases such as a DiGeorge syndrome diagnosed in 2004 and Evans Syndrome with auto-immune haemolytic anaemia diagnosed in 2006. Initially, this child was treated with an underdosing of oseltamivir, however, as the influenza-like syndrome persisted with a suspicion of viral pneumonia, led to the antiviral treatment dose review. The NA inhibition assay confirmed that this virus was resistant to oseltamivir. The NA inhibition assays performed in our viral isolates and accordingly to our sequence data, revealed that the majority of Portuguese AH1N1pdm viruses were sensitive to NAI. All neuraminidases presented the N248D substitution, except for A/Lisboa/35/2009, a viral strain from an early clade (at least, previous to clade 4) as it lacks also V106I amino acid change. Other amino acid substitutions, such as E119V, I223V, Q136K, known to reduce the susceptibility to NAI, were not found in our isolates. Conclusions: The use of conventional sequence analysis for monitoring the molecular markers of antiviral resistance in influenza A virus provides a valuable tool for an early detection of antiviral resistant strains.
- 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.
- Using clinical criteria on seasonal and pandemic influenza: what to look for?Publication . Gonçalves, Paulo; Paixão, Eleonora; Machado, Ausenda; Cordeiro, Rita; Nunes, Baltazar; Pechirra, Pedro; Guiomar, RaquelThe reliability of clinical criteria on the diagnosis of influenza has been a question which has generated much debate in the scientific community. In Portugal, Influenza-like illness (ILI) cases have been notified by two sentinel networks, one of general practitioners (“Médicos-Sentinela”) and another of emergency rooms of hospitals and health centres, to the National Influenza Reference Laboratory in the context of the National Influenza Surveillance Programme, using clinical criteria adapted from the International Classification of Health Problems in Primary Care. With the emergence of a novel influenza A(H1N1) virus circulating in humans, the clinical definition of an influenza case was reformulated to accommodate the clinical features observed at that time. But how clinically different was the new variant A(H1N1) infection when compared to the seasonal influenza? In this study we propose to evaluate the signs and symptoms present in influenza cases diagnosed in Portugal during a seasonal influenza winter with those reported during the recent A(H1N1) pandemic. Also, we intend to evaluate the clinical criteria used for the notification of influenza cases during these two periods. 2. Materials and methods ILI cases were diagnosed for influenza at the National Influenza Reference Laboratory in the context of the National Influenza Surveillance Programme during the 2008/2009 influenza winter season and 2009/2010 pandemic period, from week 38/2008 through week 20/2010. Clinical information on those cases included the presence of signs and symptoms related with ILI as defined by the International Classification on Health Problems in Primary Care and on the case definition introduced by the European Commission Decision 202/253/EC (signs/symptoms analysed: sudden onset of symptoms, presence of fever, weakness, headache, myalgia, cough, sore throat, respiratory difficulty, chills and contact with an influenza patient). The odds ratio (OR) of being positive for influenza for each sign/symptom was calculated, independently and using multivariable logistic regression with all the signs/symptoms. Results were compared with the clinical definition of influenza used during both periods. 3. Results During the 2008/2009 winter, when seasonal influenza A(H3) viruses were dominant, all signs/symptoms analysed revealed an OR associated with a risk of being positive for influenza. Those with a higher risk were cough (OR 9.5, CI95% 4.7-19.3), fever (OR 4.5, CI95% 2.8-7.1), chills (OR 2.3, CI95% 1.5-3.5), myalgia (OR 1.7, CI95% 1.1-2-7) and contact with another ILI patient (OR 1.5, CI95% 1.1-2.0). Adjusting for all the signs/symptoms, the multivariable logistic regression reveals cough (OR 10.7, CI95% 5.2-22.0), fever (OR 5.1, CI95% 3.2-8.2) and contact with another patient (OR 1.4, CI95% 1.0-1.9) to be statistically significant. For the pandemic season 2009/2010, the signs/symptoms associated with a higher risk of being positive for influenza were fever (OR 4.5, CI95% 2.5-8.3), cough (OR 3.2, CI95% 2.1-4.8) and contact with another patient (OR 2.0, CI95% 1.4-2.8). Multivariable logistic regression also indicates these signs/symptoms to be statistically significant, after adjustment. 4. Conclusions Although initial evidences that the new influenza pandemic variant A(H1N1) could cause a clinically different infection, and fears that the disease would present a more severe profile than seasonal influenza, data collected in our country through the National Influenza surveillance Programme indicates that both situations were clinically similar in their presentation. This fact has been supported by studies from other countries.
- Pandemic Influenza Virus Surveillance in Portugal: The Laboratory Network for the Diagnosis of Influenza A(H1N1)2009 InfectionPublication . Guiomar, Raquel; Pechirra, Pedro; Gonçalves, Paulo; Cordeiro, Rita; Conde, Patrícia; Arraiolos, Ana; Batista, Inês; Paixão, Eleonora; Nunes, Baltazar; Furtado (on behalf of the Laboratory Network for the Diagnosis of Influenza A(H1N1)2009 Infection), CristinaIn April 2009 a new influenza A(H1N1) virus of swine origin disseminated throughout the world, resulting in the first pandemic of the XXI century. To face the increasing number of diagnosis being requested, a National Laboratory Network for Influenza Surveillance of the new influenza A(H1N1)pandemic virus was activated in Portugal. This is a descriptive study of the Influenza-like Ilness (ILI) cases reported by this network. Association between the variables was evaluated by chi-squared test. Over 62089 ILI cases were notified, 25594 (41.2%) cases were laboratory confirmed A(H1N1)pdm virus, from week 17/2009 to week 15/2010. In the week 33 (summer) were detected 1039 (4.1%) positive cases for A(H1N1)pdm virus although the winter peak occurred in week 46 with 3131 (12.5%) A(H1N1)pdm positive cases. In the age group of 5-14 years old were detected the majority of positive cases 9983 / 15785 (63.0%) opposite in the elderly group (>65 years old) was detected the lower number of A(H1N1)pdm positive cases, 280/2361 (11.0%). The distribution by gender accounts 40.4% of female and 42.3% of male positive cases. The signs and/or symptoms present were analysed revealing that headache (49.7%), cough and myalgies (46.5%) and odinophagia (46.1%) were statistically associated with A(H1N1)pdm positive cases. The chronic pulmonary disease seemed to be more associated with laboratory confirmed A(H1N1)pdm cases. Ninety five strains were isolated and antigenically characterised, 45 were taken for genetic analysis (HA and NA gene). All the strains were antigenically and genetically like the pandemic vaccine strain. It was detected only one strain with the mutation H275Y in the neuraminidase, resistant to oseltamivir. This Laboratory network was an important tool to monitories and control the evolution of the pandemic.
- Towards a risk assessment for Giardia sp. and Cryptosporidium sp. in Portuguese fluvial beaches: a seasonal sampling over two yearsPublication . Júlio, C.; Ferreira, I.; Martins, S.; Sá, C.; Ângelo, H.; Guerreiro, J.; Tenreiro, R.Abstract background: Waterborne outbreaks of diarrhoeal illness reported worldwide are mostly associated with Cryptosporidium sp. and Giardia sp. Lake and river waters contaminated with (oo)cysts are major routes of human exposure making essential the development of preventive strategies for water safety. Since monitoring of water contamination with (oo)cysts is not routinely performed in Portugal, this study aims to unveil the possible associations between Portuguese fluvial beach characteristics and risk for public health caused by different genotypes of Giardia sp. and Cryptosporidium sp.. Abstract Methods: Nineteen beaches were selected according to land use and environmental parameters and sampled, on winter and summer, for the presence of Giardia sp. and Cryptosporidium sp., as well as faecal indicators and physicochemical parameters. Immunomagnetic separation was performed according the US EPA Method 1623 with Dynal procedure (Dynabeads), followed by detection of (oo)cysts by immunofluorecence microscopy after staining with FICT-labelled monoclonal antibody. Cysts viability was also confirmed by nucleic acid dye (DAPI) staining. Abstract Results: The results show that Giardia cysts are present at least in 83% of the sampled beaches. Presence of Cryptosporidium oocysts was not lower than 74%. Additionally, seasonal differences on (oo)cysts amount were perceived. A dendrogram analysis highlighted different clusters which evidence patterns among the sampled beaches. Principal Component Analysis also indicates distinct weights for land use, physical-chemical and microbiological parameters in these different clusters. Abstract Conclusions: The results of the present study indicate that Giardia sp. and Cryptosporidium sp. are widely distributed and should be considered as a public health issue. Moreover, beach clusters turn out to be a helpful tool to assess the public health risk.
- Occurrence of plasmid-mediated quinolone resistance among bacteria isolated in animals in PortugalPublication . Ferreira, Eugénia; Clemente, Lurdes; Jones-Dias, Daniela; Francisco, Ana Patrícia; Manageiro, Vera; Themudo, Patrícia; Albuquerque, Teresa; Amado, Alice; Caniça, ManuelaBackground: Plasmid-mediated quinolone resistance (PMQR) is increasingly identified worldwide in Enterobacteriaceae. The aim of this study was to evaluate the extension of PMQR in isolates from animals in Portugal. Methods: We screened 186 Enterobacteriaceae isolates for the presence of PMQR determinants, identified at National Laboratory of Veterinary Research (2008-2009). A total of 92 Salmonella isolates were isolated from broilers, layers and pigs, and 94 Escherichia coli were from farm animals, birds and mammals. Susceptibility testing of all isolates was performed by disk diffusion method, and MICs against nalidixic acid, ciprofloxacin, gatifloxacin, levofloxacin, ofloxacin, enrofloxacin, morbifloxacin and norfloxacin were determined by E-test for PMQR-positive isolates. PCR and nucleotide sequencing, by using specific primers, were used to screen for the presence of PMQR-encoding genes. The genetic context of PMQR genes was evaluated by using different molecular methods. Results: We identified 5 qnrC-positive isolates: 2 Salmonella enteritidis collected in 2 layer chicken and in 3 E. coli from 2 broilers and one pig; 3 qnrS1 genes were detected in E. coli isolates from a broiler (co-expressing a qnr-C gene), a dog and a turtle-dove. The aac-(6’)-Ib-cr gene was detected in an E. coli isolated from a mammalian. Seven PMQR-positive isolates showed diminished susceptibility to at least one quinolone, and one was detected in the range of susceptibility against the seven (fluoro)quinolones tested. Three E. coli and one S. enteritidis were PMQR- and TEM-1 and/or CTX-M-15-producing isolates. An E. coli with qnrC, qnrS1, and blaTEM-1 genes and an E. coli with qnrC gene were positive for genes coding to class 1 integrons. Conclusions: This survey showed that PMQR determinants are present in animals from different environments in Portugal, including food-producing animals, with a high frequency (3%) of QnrC-producing isolates. Susceptibility results demonstrate the difficulty to predict the PMQR mechanisms by phenotypic methods. Overall, the study suggests that PMQR genes are undergoing a dissemination process, which needs surveillance.
- Epidemiology of plasmid-mediated quinolone resistance determinants in bacterial isolates from animals and foods with co-resistance to several antibioticsPublication . Ferreira, Eugénia; Francisco, Ana Patrícia; Jones-Dias, Daniela; Manageiro, Vera; Caniça, ManuelaBackground: The use of (fluoro)quinolones both in humans and animals has contributed to the selection of resistant bacteria, limiting the agents available for treatment. This study aims to search for plasmid-mediated quinolone resistance (PMQR) determinants to give information about these expanding resistance mechanisms, their capacity of dissemination among different bacteria by mobile elements, and the role that they play in facilitating co-resistance to several antimicrobials. Methods: In this work we investigated a collection of Escherichia coli (n=183) and Salmonella spp (n=186) isolates recovered from several types of consumable meat (n=93) and from samples collected from food-producing animals received from diagnostic (n=276). Antibiotic susceptibility testing against different classes of antibiotics was performed by disk diffusion and MICs were determined by E-test for PMQR-positive isolates. Molecular characterization of the isolates included PCR detection of PMQR determinants and of beta-lactamase-encoding genes (for putative producing isolates with diminished susceptibility to beta-lactams); the respective coding proteins were identified through nucleotide sequencing. Class 1 integrons (int1 gene) were searched by PCR. Results: Overall, we detected 19/369 PMQR determinants, namely 11 QnrS1 in E. coli isolates from poultry. Two of these strains also carried TEM-1 beta-lactamase and one of them carried TEM-135 plus SHV-108 (ESBLs). Among the 6 QnrB detected, we identified 3 QnrB19: one in an E. coli (from a pig) and 2 in Salmonella spp isolates (from poultry), and 3 QnrB2 in Salmonella spp isolates (from embryonated eggs). We also have detected 2 Aac-(6’)-Ib-cr in E. coli and Salmonella spp isolates, from broiler and bovine origin, respectively. One isolate carrying Aac-(6’)-Ib-cr was from consumable meat. Among PMQR-negative isolates, we detected one DHA-type (plasmid-mediated AmpC β-lactamase) Salmonella spp-producing isolate, as well as one SHV-12 and one TEM-135 (ESBLs) E. coli-producing isolates. Class 1 integrons were detected among 8 out of 19 PMQR-positive isolates, but were not always associated to multidrug-resistance (MDR). Conclusions: This investigation has demonstrated that PMQR may act additively with other plasmid-encoded resistance mechanisms in 16% of studied isolates and in 42% may be disseminated by plasmids carrying class 1 integrons, contributing to the scenario of MDR in animal production facilities.
- Rab8 is required for phagocytosis and phagosomal maturationPublication . Jordão, Luísa; Ramalho, Jose; Seabra, Miguel; Vieira, Otilia V.