Browsing by Author "Cunha, Mário"
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- Characterization of the Human Papillomavirus 16 Oncogenes in K14HPV16 Mice: Sublineage A1 Drives Multi-Organ CarcinogenesisPublication . Cochicho, Daniela; Nunes, Alexandra; Gomes, João Paulo; Martins, Luís; Cunha, Mário; Medeiros-Fonseca, Beatriz; Oliveira, Paula; Bastos, Margarida M.S.M.; Medeiros, Rui; Mendonça, Joana; Vieira, Luis; Gil da Costa, Rui M.; Felix, AnaThe study of ()-induced carcinogenesis uses multiple in vivo mouse models, one of which relies on the cytokeratin 14 gene promoter to drive the expression of all HPV early oncogenes. This study aimed to determine the HPV16 variant and sublineage present in the K14HPV16 mouse model. This information can be considered of great importance to further enhance this K14HPV16 model as an essential research tool and optimize its use for basic and translational studies. Our study evaluated HPV DNA from 17 samples isolated from 4 animals, both wild-type (n = 2) and HPV16-transgenic mice (n = 2). Total DNA was extracted from tissues and the detection of HPV16 was performed using a qPCR multiplex. HPV16-positive samples were subsequently whole-genome sequenced by next-generation sequencing techniques. The phylogenetic positioning clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). A comparative genome analysis of K14HPV16 samples revealed three mutations to the human papillomaviruses type 16 sublineage A1 representative strain. Knowledge of the HPV 16 variant is fundamental, and these findings will allow the rational use of this animal model to explore the role of the A1 sublineage in HPV-driven cancer.
- Collaborative study Brazil-Portugal - alidation of HPV lyophilized samples for the control of molecular testsPublication . Menezes, Maria Elizabeth; Fedrizzi, Edson Natal; Correa, José Abol; Cochicho, Daniela; Martins, Luís; Cunha, Mário; Ornelas, Carmo; Verdasca, Nuno; Faria, Ana PaulaThere are about 120 types of Human Papilloma Virus (HPV). The major importance about it is the oncogenic potential of some types. They are referred as a high and low risk for oncogenic potential. Given the importance of the disease it causes, and the use of different laboratorial techniques, mainly techniques of molecular biology, it is mandatory to have an External Quality Assurance (EQA) program base on harmonized standards that rely on consistent controls. The participation in EQA’s programs is mandatory in laboratorieswith an ISO 15189/17025 accreditation implemented. The control of Molecular Biology techniques is absolutely necessary for the quality assurance of the results, the tracking and monitorization of the performance of the reagents. However, one of its limitations is the stability, homogeneity of the material, (ISO/IEC 17043:2010 requisite) as well as the availability of control samples adequate for the parameter to be analyzed. The PNCQ, in order to fill this gap, lyophilised the HPV samples control. In order to do the validation and to test the reproducibility of these lyophilized samples, PNCQ sent to IBIOTECNO , INSA-PNAEQ -DDI and the IPOLFG-SPCLV to be analysed by different methods and reagents in two different countries and different laboratories.
- Collaborative study Brazil-Portugal: validation of HPV lyophilized samplesPublication . Menezes, Maria Elizabeth; Tourinho, Frederico; Abol Corrêa, José; Faria, Ana Paula; Cunha, MárioHPV is an important virus that is responsible for more than 99% of cancer in women. Preparation and validation of lyophilized samples to use as a control of molecular biology tests for HPV is very important. There are several techiniques to performer molecular test to detecte HPV however, it is necessary that their is a sample control whith can be used independent of methodology. Validation of lyophilized samples by different molecular techniques to have harmonized standards relies on consistent control.
- 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.
- Epidemiology and genetic variability of respiratory syncytial virus in Portugal, 2014-2018Publication . Sáez-López, Emma; Cristóvão, Paula; Costa, Inês; Pechirra, Pedro; Conde, Patrícia; Guiomar, Raquel; Peres, Maria João; Viseu, Regina; Lopes, Paulo; Soares, Vânia; Vale, Fátima; Fonseca, Patricia; Freitas, Ludivina; Alves, Jose; Pessanha, Maria Ana; Toscano, Cristina; Mota-Vieira, Luísa; Veloso, Rita Cabral; Côrte-Real, Rita; Branquinho, Paula; Pereira‑Vaz, João; Rodrigues, Fernando; Cunha, Mário; Martins, Luís; Mota, Paula; Couto, Ana Rita; Bruges-Armas, Jácome; Almeida, Sofia; Rodrigues, Débora; Portuguese Laboratory Network for the Diagnosis of Influenza InfectionIntroduction: 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.
- Influenza seroprotection correlates with predominant circulating viruses during 2014/15 and 2015/16 seasons in PortugalPublication . Guiomar, Raquel; Cristóvão, Paula; Conde, Patrícia; Costa, Inês; Pechirra, Pedro; Rodrigues, Ana Paula; Pereira da Silva, Susana; Nunes, Baltazar; Mouro Pinto, Rita; Sobrinho Simões, Joana; Costa, Maria do Rosário; Guimarães, João Tiago; Rodrigues, Fernando; Correia, Lurdes; Pereira-Vaz, João; Caseiro, Paula; Cabral Veloso, Rita; Mota Vieira, Luísa; Pimentel Couto, Ana Rita; Santos, Margarida; Bruges Armas, Jácome; Branquinho, Paula; Corte-Real, Rita; Martins, Luís; Cunha, Mário; Almeida, Sofia; Viseu, Regina; Inácio, Filipe; Peres, Maria João; Milho, Luís; Fernandes, Aida; Maurílio, Manuel; Caldeira, Filomena; Sanches, Raquel; Dantas, Filipa; Freitas, Ludivina; Andrade, Graça; Mota, PaulaBACKGROUND: Population immune profile for influenza is highly affected by circulating influenza viruses, thus changing the risk of infection for influenza. This study aims to assess influenza immunity in the Portuguese population by age groups, during 2014 and 2015 and establish a relationship between seroprotection and circulating influenza viruses in 2014/15 and 2015/16 seasons. METHODS: Two cross-sectional studies were developed based on a convenience serum sample collected in June 2014 (n=626) and July 2015 (n=675) in hospitals from mainland and Azores and Madeira.Serums equally represent all age groups. Antibody titers were evaluated by HI assay for strains recommended for seasonal influenza vaccine northern hemisphere,2014/15 and 2015/2016. Seroprevalences were estimated for each strain by age group and the association with seasonal cumulative influenza-like illness (ILI) rates for influenza virus during both seasons was analised. RESULTS: In June 2014 the highest seroprotection was observed for influenza A(H3) (39.0%; 95% CI: 36.2-43.8%) and A(H1)pdm09 (29.7; 95% CI: 26.3-33.4%), with higher levels in children 5-14 years old. In 2014/2015 a dominant circulation of influenza B/Yamagata was observed with high incidence rates in individuals under 65 years old, the ones that had lower seroprotection. Although before the start of the season high protection for A(H3) was observed, the circulation of the new drift A(H3) strains had gained an immunological advantage,in accordance with A(H3) elevated incidence rates observed during 2014/15. In July 2015 the highest seroprotection was observed for influenza B/ Yamagata (55.1%; 95% CI: 51.4-58.9%), 2.4 times the estimated 2014.This increase was even more pronounced in younger (≤ 4 years old), 6.3 times increase in 2015.This fact is in agreement with the predominant influenza B virus detected and the high ILI incidence rate observed in children during 2014/2015 epidemic. Seroprotection levels for influenza A in July 2015 were not significantly different from 2014.During 2015/16 season, influenza A(H1N1)pdm09 was predominant, with high incidence rate in < 65 year old. Influenza B/Victoria lineage,although detected at low levels increased in frequency, in agreement with the lowest level of seroprotection detected in the general population before the start of 2015/2016 season (21.8%; 95% CI: 18.7-24.0%). CONCLUSIONS There was a correlation between virus circulation, incidence rates for each age group and the previous seroprotection for seasonal influenza viruses.Our study highlights the value of measuring the serological profile for influenza to establishe risk groups for infection for which an increase preventive measures, including vaccination, should be fostered.
- Influenza severe cases in hospitals, between 2014 and 2016 in PortugalPublication . Guiomar, Raquel; Pechirra, Pedro; Cristóvão, Paula; Costa, Inês; Conde, Patrícia; Corte-Real, Rita; Branquinho, Paula; Silvestre, Maria José; Almeida Santos, Madalena; Fernandes, Isabel; Dias, Isabel; Rodrigues, Sónia; Sena, Nadir; Lazzara, Daniela; Sobrinho Simões, Joana; Costa, Maria do Rosário; Guimarães, João Tiago; Rodrigues, Fernando; Pereira-Vaz, João; Correia, Lurdes; Andrade, Graça; Freitas, Ludivina; Figueira, Neuza; Sanches, Raquel; Marques, Mónica; Barros, Margarida; Mota Vieira, Luísa; Cabral Veloso, Rita; Castelo Branco, Cláudia; Pimentel, Sílvia; Duarte, Joana; Pereirinha, Tânia; Bulhões, Sara; Moniz, Raquel; Brilhante, Maria José; Bruges Armas, Jácome; Pimentel Couto, Ana Rita; Santos, Margarida; Soares, Marta; Melo Cristino, José; Ribeiro, Carlos; Carvalho, Dinah; Barreto, Rosário; Ramos, Maria Helena; Castro, Ana Paula; Matos Santos, Ana Cláudia; Cunha, Mário; Martins, Luís; Almeida, Sofia; Peres, Maria João; Viseu, Regina; Inácio, Filipe; Mota, PaulaBackground: Since 2009, the Portuguese Laboratory Network (PLNID) for Influenza Diagnosis has integrated 15 Laboratories in mainland and Atlantic Islands of Azores and Madeira. This PLNID added an important contribute to the National Influenza Surveillance Program regarding severe and hospitalized influenza cases. The present study aims to describe influenza viruses detected in influenza like illness (ILI) cases: outpatients (Outp), hospitalized (Hosp), and intensive care units (ICU), between 2014 and 2016. Methods: The PLNID performs influenza virus diagnosis by biomolecular methodologies. Weekly reports to the National Influenza Reference Laboratory ILI cases tested for influenza. Reports include data on detecting viruses, hospital assistance, antiviral therapeutics, and information on death outcome. Were reported during two winter seasons 8059 ILI cases,being 3560 cases in 2014/15 (1024 in Outp, 1750 Hosp, and 606 in ICU) and 4499 cases in 2015/2016 (1933 in Outp, 1826 Hosp, and 740 in ICU). Results: The higher percentage of influenza positive cases were detected in Outp in both seasons, 18% during 2014/15 and 20% in 2015/16. In 2014/15,influenza cases were more frequent in individuals older than 65 years old and these required more hospitalizations,even in ICU. In 2015/16,the influenza cases were mainly detected in individuals between 15-64 years old. A higher proportion of influenza positive cases with hospitalization in ICU were observed in adults between 45-64 years old.During the study period,the predominant circulating influenza viruses were different in the two seasons: influenza B and A(H3) co-circulated in 2014/15,and influenza A(H1)pdm09 was predominant during 2015/16. Even when influenza A is notthe dominant virus, A(H3) and A(H1)pdm09 subtypes correlate with higher detection rate in hospitalized cases (Hosp and UCI), with higher frequencies in adults older than 45. Influenza B,detected in higher proportion in outpatients, was frequently relatedwith influenza cases in younger age groups: 0-4 and 5-14 years old. Conclusions: This study highlights the correlation of theinfluenza virus type/subtype that circulates in each season with the possible need for hospitalization and intensive care in special groups of the population. Circulation of influenza A subtypes can cause more frequentdisease in individuals older than 45, with need of hospitalization including intensive care. On the other hand, influenza B is more frequently associated with less severe cases and with infection in children and younger adults. Influenza B circulation might predict lower number of hospitalizations.The identification of influenza type in circulation,byPLNID ineach season, could guide action planning measures in population health care.
- Influenza virus type/subtype and different infection profiles by age group during 2017/2018 seasonPublication . Guiomar, Raquel; Pechirra, Pedro; Cristóvão, Paula; Costa, Inês; Conde, Patrícia; Côrte-Real, Rita; Branquinho, Paula; Garcia, David; Conde, Sílvia; Rodrigues, Fernando; Pereira-Vaz, João; Alves, José; Freitas, Ludivina; Mota Vieira, Luísa; Cabral Veloso, Rita; Bruges Armas, Jácome; Couto, Ana Rita; Ribeiro, Carlos; Barreto, Rosário; Cunha, Mário; Martins, Luís; Almeida, Sofia; Peres, Maria João; Viseu, Regina; Mota, Paula; Lopes, Paulo; Soares, Vânia; Vale, Fátima; Fonseca, Patrícia; Toscano, Cristina; Dias, AnaBackground: Influenza has a major impact in hospitalization during each influenza season. We analysed the influenza type/subtype distribution by age group and medical care wards (ambulatory, hospital, intensive care unit). Material and Methods: During 2017/2018 season, 14 hospitals from Portugal mainland and Atlantic Island (Azores and Madeira) reported to the National Influenza Centre 13747 cases of respiratory infection, all tested for influenza type and/or subtype. Epidemiological data: age, sample collection, hospital dwelling service and patient outcome were reported. Results: From the 13747 reported cases, 3717(27%) were influenza positive of which 2033 (55%) were influenza B, 722 (19%) A unsubtyped, 505 (14%) AH3, 442 (12%) AH1pdm09 and 15(0,1%) mixed infections. Influenza A was detected in 71% (204/208) of toddlers(<5 years) although in the remaining age groups influenza B was detected in more than 50% of the confirmed flu cases. Influenza B was the predominant virus in hospitalized and ICU influenza cases between 5-14 years (69% and 75%, respectively) and played a major role in elderly (65+ years) hospitalized and ICU cases(57% and 67%, respectively). AH1pdm09 virus was detected in 30% of the influenza confirmed ICU patients, 2.1 times more than in hospitalized cases in other wards and 3.3 times more than influenza AH1pdm09 cases in ambulatory care. Influenza mixed infection were detected sporadically,mainly in hospitalized and ICU patients. From 2080 known outcomes, 40(1.9%) patients deceased, influenza was confirmed in 11(28%) of these cases. Conclusions: Cocirculation of different influenza virus type/subtype may indicate different infection profiles by age groups and should guide influenza preventive/treatment measures.
- Long-Term Evolution of SARS-CoV-2 in an Immunocompromised Patient with Non-Hodgkin LymphomaPublication . Borges, Vítor; Isidro, Joana; Cunha, Mário; Cochicho, Daniela; Martins, Luís; Banha, Luís; Figueiredo, Margarida; Rebelo, Leonor; Trindade, Maria Céu; Duarte, Sílvia; Vieira, Luís; Alves, Maria João; Costa, Inês; Guiomar, Raquel; Santos, Madalena; Cortê-Real, Rita; Dias, André; Póvoas, Diana; Cabo, João; Figueiredo, Carlos; Manata, Maria José; Maltez, Fernando; Gomes da Silva, Maria; Gomes, João PauloRecent studies have shown that persistent SARS-CoV-2 infections in immunocompromised patients can trigger the accumulation of an unusual high number of mutations with potential relevance at both biological and epidemiological levels. Here, we report a case of an immunocompromised patient (non-Hodgkin lymphoma patient under immunosuppressive therapy) with a persistent SARS-CoV-2 infection (marked by intermittent positivity) over at least 6 months. Viral genome sequencing was performed at days 1, 164, and 171 to evaluate SARS-CoV-2 evolution. Among the 15 single-nucleotide polymorphisms (SNPs) (11 leading to amino acid alterations) and 3 deletions accumulated during this long-term infection, four amino acid changes (V3G, S50L, N87S, and A222V) and two deletions (18-30del and 141-144del) occurred in the virus Spike protein. Although no convalescent plasma therapy was administered, some of the detected mutations have been independently reported in other chronically infected individuals, which supports a scenario of convergent adaptive evolution. This study shows that it is of the utmost relevance to monitor the SARS-CoV-2 evolution in immunocompromised individuals, not only to identify novel potentially adaptive mutations, but also to mitigate the risk of introducing "hyper-evolved" variants in the community.