Browsing by Author "Ferreira, Teresa"
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- Aspergillus section Fumigati – Epidemiological trends - A perspective from a National Reference LaboratoryPublication . Sabino, Raquel; Simões, Helena; Francisco, Mariana; Viegas, Carla; Toscano, Cristina; Batista, JuditeTeresa; Ferreira, Teresa; Veríssimo, CristinaObjectives: Aspergillus fumigatus is the most frequent agent of aspergilosis and reports on infections caused by this species or its siblings are becoming more frequent, together with the increasing number of at risk patients. Nowadays, due to the rising concerns on emerging antifungal resistance, the epidemiological surveillance for clinical and environmental isolates is mandatory. The overall objective of the project is to understand the epidemiology of the Aspergillus isolates (species and antifungal resistance) collected in the Portuguese National Reference Laboratory through our surveillance system on Aspergillus. Methods: During the period 2013-2017, 117 Aspergillus section Fumigati isolates were collected at the National Health Reference Dr. Ricardo Jorge, through the surveillance system on Aspergillus. Isolates were obtained from different patient samples from 15 healthcare institutions of all country, and from different environmental sources (air or surfaces sampling). All isolates were plated for growth as single colonies on malt extract agar with chloramphenicol. These isolates were identified on the basis of macro and microscopic morphology and through the use of molecular tools. Genomic DNA was prepared from each isolate and the sequencing of the Internal Transcribed Spacers (ITS) regions as well as of the gene codifying to calmodulin. Surveillance of azole resistance was performed firstly using Sabouraud dextrose agar supplemented with itraconazole (ICZ), voriconazole (VCZ), and posaconazole (PCZ). When growth was observed, the minimal inhibitory concentration (MIC) was determined by broth microdilution method. In case of doubt, a specific PCR for detection of mutations in the Cyp51A gene of A. fumigatus was performed using the AsperGenius® multiplex real-time PCR assay. Results: From the isolates collected during the study period, 94 were from clinical (human) sources, 2 from animals diagnosed with aspergillosis and 21 from environmental sources Clinical isolates were obtained from 90 patients (53 males, 34 females, 3 not known), with ages ranging from 37 days to 88 years old. Most of these isolates (98%) were from respiratory specimens. The underlying diseases reported are, among others, cystic fibrosis, COPD, HIV, asthma, and neoplasms. In total, 111 A. fumigatus sensu stricto isolates were identified, followed by 3 A. lentulus, 2 A. felis and 1 A. hiratsukae (from hospital environment). In 7 cases, the morphological identification did not matched with the correct species-section. Interestingly, the 5 clinical cryptic species were from the same hospital. Regarding susceptibility, relevant and residual growths were obtained in azole resistance screening media (Table 1). The positive results were then screened by microdilutions and by detection of Cyp51A mutations and resistances were not confirmed. Conclusions: The understanding of local resistance patterns is valuable to assess shifts in the epidemiology of Aspergillus (and therefore, to manage therapeutic approaches). In our collection of Fumigati isolates, 5% of them were cryptic species. Although we did not confirm azole resistance by microdilution or detection of Cyp51A mutations, the MIC values obtained suggest that the median values are higher than what is described in other studies (1.4 to ICZ, 0.4 to PCZ),which may explain the growth in screening media and may suggest a local epidemiology.
- Biomarkers and genetic modulators of cerebral vasculopathy in sub-Saharan ancestry children with sickle cell anemiaPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Ferreira, Emanuel; Mendonça, Joana; Vieira, Luís; Maia, Raquel; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Silva, Rita; Kjollerstrom, Paula; Faustino, PaulaWe investigated biomarkers and genetic modulators of the cerebral vasculopathy (CV) subphenotype in pediatric sickle cell anemia (SCA) patients of sub-Saharan African ancestry. We found that one VCAM1 promoter haplotype (haplotype 7) and VCAM1 single nucleotide variant rs1409419_T were associated with stroke events, stroke risk, as measured by time-averaged mean of maximum velocity in the middle cerebral artery, and with high serum levels of the hemolysis biomarker lactate dehydrogenase. Furthermore, VCAM-1 ligand coding gene ITGA4 variants rs113276800_A and rs3770138_T showed a positive association with stroke events. An additional positive relationship between a genetic variant and stroke risk was observed for ENPP1 rs1044498_A. Conversely, NOS3 variants were negatively associated with silent cerebral infarct events (VNTR 4b_allele and haplotype V) and CV globally (haplotype VII). The -alpha3.7kb–thal deletion did not show association with CV.However, it was associated with higher red blood cell and neutrophil counts, and lower mean corpuscular volume, mean corpuscular hemoglobin and red cell distribution width. Our results underline the importance of genetic modulators of the CV sub-phenotype and their potential as SCA therapeutic targets. We also propose that a biomarker panel comprising biochemical, hematological, imaging and genetic data would be instrumental for CV prediction, and prevention.
- Endothelial factors and stroke risk in pediatric sickle cell anemia patients: insights from VCAM1 and ITGA4 variantsPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rute; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle cell anemia (SCA) arises from homozygosity for the mutation c.20A>T in the HBB gene which originates hemoglobin S (HbS). In hypoxic conditions, HbS polymerizes inside erythrocytes deforming them and ultimately leading to hemolysis and vaso-occlusion. SCA shows a multifactorial-like behaviour with a high heterogeneity of clinical features, with stroke being the most severe of them. This heterogeneity may arise from underlying genetic modifiers, namely those affecting vascular adhesion/endothelial dysfunction. These include genes encoding the VCAM-1 molecule and its ligand VLA-4 (ITGA4 or integrin α4), increasingly studied due to their expression in activated human endothelium and leucocytes/stress reticulocytes, respectively. The aim of this study was to identify putative genetic modulators of stroke risk by analyzing 70 pediatric SCA patients, grouped according to their degree of cerebral vasculopathy. Molecular analysis was performed using Next-Generation Sequencing (NGS) and Sanger Sequencing. R software was used for statistical analyses and association studies. In silico studies were performed using PHASE, TFbind, PROMO and Human Splicing Finder software tools. We identified six different VCAM1 promoter variants and seven haplotypes. The VCAM1 promoter rs1409419_T allele was associated with stroke events (p=0.008; O.R.= 4.33; C.I.95% =1.391-14.257), while one VCAM1 promoter haplotype was found to be protective of stroke (p=0.011; O.R.=0.22; C.I.95% =0.048-0.784). On the ITGA4 gene, forty variants were found, six of them novel. All patients presented with at least one variant in this gene. We observed co-inheritance of specific sets of ITGA4 variants indicating the presence of haplotypes not previously described. Additionally the presence of specific variants seems to result in a predisposition for either high reticulocyte count, elevated lactate dehydrogenase, raised bilirubin levels or increased transcranial Doppler velocity values. Our results reinforce the role of endothelial molecules and blood cell interaction in SCA severity. The association between specific VCAM1, as well as ITGA4, variants with certain cerebral vasculopathy predictors, further enhances their putative modulating effect on pediatric stroke severity and prognosis. These findings provide additional clues on the SCA pathophysiology and uncover features of both genes that may prove to be crucial as potential therapeutic targets.
- Etiologia das infeções fúngicas invasivas e subcutâneas: análise dos dados da Rede Nacional de Vigilância Laboratorial das Infeções Fúngicas Invasivas e Subcutâneas (IFIs), 2013-2018Publication . Veríssimo, Cristina; Toscano, Cristina; Ferreira, Teresa; Abreu, Gabriela; Simões, Helena; Diogo, José; Queirós, Ana Maria; Santiago, Felicidade; Lima, Ana; Sabino, RaquelA epidemiologia das infeções fúngicas invasivas tem vindo a alterar-se com o surgimento de novos agentes etiológicos. A Rede Nacional de Vigilância Laboratorial de Infeções Fúngicas Invasivas e Subcutâneas (IFI) teve início em 2013 com o objetivo de melhor compreender a epidemiologia destas infeções em Portugal. O objetivo deste estudo é analisar os dados obtidos através da Rede entre junho de 2013 e setembro de 2018. Os laboratórios participantes asseguram a comunicação dos casos de IFIs ao Laboratório Nacional de Referência do INSA, enviando amostras biológicas ou isolados, acompanhados de um inquérito contendo informações demográficas, laboratoriais e avaliações clínicas dos doentes com suspeita de infeção fúngica. O estudo incluiu: i) casos de infeção fúngica invasiva (IFI) por fungos filamentosos/dimórficos, ii) casos de IFI provável, de acordo com os critérios estabelecidos pelo EORTC/MSG 2008, e iii) casos de infeções fúngicas subcutâneas. As infeções por leveduras não foram incluídas. Entre junho de 2013 e setembro de 2018, foram incluídos 52 casos, distribuídos da seguinte forma: IFI comprovada (por fungos filamentosos) (n=9); Infeção fúngica subcutânea (n=17); Infeção por fungos dimórficos endémicos (n=9), totalizando 67% de casos de IFI comprovados (n=35) e 33% de casos IFI provável (n=17). Os dados obtidos chamam a atenção para a grande diversidade de espécies envolvidas em infeções fúngicas profundas, com implicações para o diagnóstico clínico/laboratorial bem como para o tratamento destas infeções
- Fetal hemoglobin level and stroke risk in children with sickle cell anemiaPublication . Nicolau, Marta; Vargas, Sofia; Coelho, Andreia; Silva, Marisa; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rute; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle Cell Anemia (SCA) is a hereditary anemia caused by a missense mutation in HBB and it is characterized by chronic hemolysis, recurrent episodes of vaso-occlusion and infection. Cerebral vasculopathy is one of the most devastating complications of the disease and even young children with SCA have a high risk of stroke. It is known that both environmental and genetic determinants are able to modulate the onset, course and outcome of the disease. Among those, the level of fetal hemoglobin (HbF) has been proposed as the most significant disease modulator. Thus, in this work, we aimed to investigate if the level of HbF in SCA children is related with the risk of stroke and if it is modulated by variants in genes, such as HBG2, BCL11A, HBS1L-MYB, and KLF1. Sixty-seven children (3 years of age) with SCA were enrolled in this study. Hematological and imaging data were retrospectively obtained from patients’ medical records at Greater Lisbon area hospitals. Patients were grouped according to their degree of cerebral vasculopathy evaluated by transcranial Doppler velocities and magnetic resonance imaging. Molecular analyses were performed using Next-Generation Sequencing, Sanger sequencing and PCR-RFLP. In silico studies and statistical analyses were done using the PolyPhen-2 and SPSS softwares, respectively. The association studies revealed that low HbF levels were associated with stroke events in SCA children (p=0.005). At the molecular level, it was observed that patients with the rarest genotypes in HBG2 (rs7482144_TT+TC) presented higher levels of HbF (p=0.031). Additionally, the rs11886868_C and the rs4671393_A alleles in BCL11A also seemed to predispose to higher HbF levels. Moreover, eleven distinct variants in KLF1 were detected (one of them novel, the p.Q342H) with 83% of the patients having at least one variant in this gene. The group of patients who have co-inherited the above mentioned variants in HBG2 and BCL11A together with at least one KLF1 variant presented the highest HbF levels (p=0.021). Our results corroborate previous studies suggesting that a low level of HbF in SCA patients is a risk factor for stroke. Furthermore, we report for the first time the importance of KLF1 variants in combination with other genetic modifiers to the final phenotypic expression of HbF in SCA children with different degrees of cerebral vasculopathy. Consequently, this study allowed the delineation of a genetic pattern with prognostic value for SCA.
- Genetic Modulation of Cerebral Vasculopathy in Children with Sickle Cell AnemiaPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rita; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle cell anemia (SCA) arises from homozygosity for the mutation c.20A>T in the HBB gene which originates hemoglobin S (HbS). In hypoxic conditions, HbS polymerizes inside erythrocytes deforming them and ultimately leading to hemolysis and vaso-occlusion. SCA shows a multifactorial-like behaviour with a high heterogeneity of clinical features, with stroke being the most severe of them. This heterogeneity may arise from underlying genetic modifiers, namely those affecting vascular hemostasis. These include genes like the ones encoding VCAM-1 and its ligand integrin α4 (expressed in activated human endothelium and leucocytes/stress reticulocytes, respectively), but also eNOS (expressed in human endothelium and regulating vascular tone). The aim of this study was to identify putative genetic modulators of stroke risk by analyzing 70 pediatric SCA patients, grouped according to their degree of cerebral vasculopathy. Molecular analysis was performed using Next-Generation Sequencing (NGS) and Sanger Sequencing. R software was used for statistical analyses and association studies. In silico studies were performed using PHASE, TFbind, PROMO and Human Splicing Finder software tools. We identified six different VCAM1 promoter variants and seven haplotypes. The VCAM1 promoter rs1409419_T allele was associated with stroke events, while one VCAM1 promoter haplotype was found to be protective of stroke. In the ITGA4 gene, forty variants were found, six of them novel. All patients presented with at least one variant in this gene. We observed co-inheritance of specific sets of ITGA4 variants indicating the presence of haplotypes not previously described. Three NOS3 variants were analysed and seven haplotypes were identified. The NOS3 promoter rs2070744_C allele was associated with stroke events, while the intron 4 VNTR 27bp_4a allele was found to be in association with risk of stroke. Our results reinforce the role of endothelial molecules and blood cell interaction in SCA severity. The association between specific variants in VCAM1 and ITGA4, as well as in NOS3, with certain cerebral vasculopathy predictors further enhances their putative modulating effect on pediatric stroke severity and prognosis. These findings provide additional clues on the SCA pathophysiology and uncover features of both genes that may prove to be crucial as potential therapeutic targets.
- Genetic modulation of stroke in children with sickle cell anaemiaPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rita; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle cell anaemia (SCA) is an autosomal recessive genetic disease that leads to the synthesis of haemoglobin S (HbS). The pathophysiology of the disease is centred on HbS polymerization inside the red blood cells, which become sickle-shaped (SSRBCs), rigid, viscous and adherent-prone to the vascular endothelium, favouring the occurrence of chronic haemolysis and vaso-occlusion. The main vascular problems of SCA arise from several pathways including endothelial dysfunction and nitric oxide (NO) metabolism. Children with SCA have a much higher risk (11% by age 20 years) of developing stroke or silent cerebral infarcts (up to 37%) than the general paediatric population. Abnormal interactions between SSRBCs and the cerebral arterial endothelium lead to endothelial injury, vaso-occlusion and tissue ischemia and result in cerebral vasculopathy (CVA) through a yet unknown pathophysiological mechanism. Current risk screening strategies rely mainly on imaging techniques (transcranial Doppler ultrasonography and magnetic resonance imaging) and children with altered results undergo regular blood transfusion and/or hydroxyurea therapy to reduce stroke risk/recurrence. However, we need more specific/sensitive biomarkers for stroke prediction/prognosis. Genetic modulators may be paramount in SCA pathophysiology and in CVA severity. They include variants in VCAM1 (endothelial dysfunction), ITGA4 (cell-cell adhesion), and NOS3 (nitric oxide metabolism. The main goals of this work are: a) improve the knowledge on the genetic architecture of paediatric cerebral vasculopathy in SCA; b) assessing the consequences of those genetic variants on gene expression/protein function; c) identify genotypic/phenotypic markers of SCA sub-phenotypes; and d) analyse their potential as genetic modulators of disease severity. This would be crucial in assessing potential pharmacological targets specifically aimed to the vascular system and instrumental for the design of novel preventive, prophylactic or therapeutic strategies.
- Genetic modulators of fetal hemoglobin expression and ischemic stroke occurrence in African descendant children with sickle cell anemiaPublication . Nicolau, Marta; Vargas, Sofia; Silva, Marisa; Coelho, Andreia; Ferreira, Emanuel; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rita; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle cell anemia (SCA) is an autosomal recessive monogenic disease with significant clinical variability. Cerebrovascular disease, particularly ischemic stroke, is one of the most severe complications of SCA in children. This study aimed to investigate the influence of genetic variants on the levels of fetal hemoglobin (Hb F) and biochemical parameters related with chronic hemolysis, as well as on ischemic stroke risk, in ninety-one unrelated SCA patients, children of sub-Saharan progenitors. Our results show that a higher Hb F level has an inverse relationship with the occurrence of stroke, since the group of patients who suffered stroke presents a significantly lower mean Hb F level (5.34 ± 4.57% versus 9.36 ± 6.48%; p = 0.024). Furthermore, the co-inheritance of alpha-thalassemia improves the chronic hemolytic pattern, evidenced by a decreased reticulocyte count (8.61 ± 3.58% versus 12.85 ± 4.71%; p < 0.001). In addition, our findings have confirmed the importance of HBG2 and BCL11A loci in the regulation of Hb F expression in sub-Saharan African SCA patients, as rs7482144_A, rs11886868_C, and rs4671393_A alleles are significantly associated with a considerable increase in Hb F levels (p = 0.019, p = 0.026, and p = 0.028, respectively). Concerning KLF1, twelve different variants were identified, two of them novel. Seventy-three patients (80.2%) presented at least one variant in this gene. However, no correlation was observed between the presence of these variants and Hb F level, severity of hemolysis, or stroke occurrence, which is consistent with their in silico-predicted minor functional consequences. Thus, we conclude that the prevalence of functional KLF1 variants in a sub-Saharan African background does not seem to be relevant to SCA clinical modulation.
- Hemorheological alterations in sickle cell anemia and their clinical consequences – the role of genetic modulatorsPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Maia, Raquel; Kjollerstrom, Paula; Lavinha, João; Faustino, PaulaSickle cell anemia (SCA) is an autosomal recessive disease caused by the HBB:c.20A>T mutation that leads to hemoglobin S synthesis. The disease presents with high clinical heterogeneity characterized by chronic hemolysis, recurrent episodes of vaso-oclusion and infection. This work aimed to characterize by in silico studies some genetic modulators of severe hemolysis and stroke risk in children with SCA, and understand their consequences at the hemorheological level. Association studies were performed between hemolysis biomarkers as well as the degree of cerebral vasculopathy and the inheritance of several polymorphic regions in genes related with vascular cell adhesion and vascular tonus in pediatric SCA patients. In silico tools (e.g. MatInspector) were applied to investigate the main variant consequences. Variants in vascular adhesionmolecule-1 (VCAM1) gene promoter and endothelial nitric oxide synthase (NOS3) gene were significantly associated with higher degree of hemolysis and stroke events. They potentially modify transcription factor binding sites (e.g. VCAM1 rs1409419 T allele may lead to an EVI1 gain) or disturb the corresponding protein structure/function. Our findings emphasize the relevance of genetic variation inmodulating the disease severity due to their effect on gene expression or modification of protein biological activities related with sickled erythrocyte/endothelial interactions and consequent hemorheological abnormalities.
- Hemorheological alterations in sickle cell anemia and their clinical consequences: the role of genetic modulatorsPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Maia, Raquel; Kjollerstrom, Paula; Lavinha, João; Faustino, PaulaSickle cell anemia (SCA) is an autosomal recessive chronic hemolytic anemia, caused by homozygosity for the HBB:c.20A>T mutation. The disease presents with high clinical heterogeneity, stroke being the most devastating manifestation. This study aimed to identify genetic modulators of severe hemolysis and stroke risk in children with SCA, as well as understand their consequences at the hemorheological level. Sixty-six children with SCA were categorised according to their degree of cerebral vasculopathy (Stroke/Risk/Control). Relevant data were collected from patients’ medical records. Several polymorphic regions in genes related to vascular cell adhesion and tonus were characterized by molecular methodologies. Data analyses were performed using R software. Several in silico tools (e.g. TFBind, MatInspector) were applied to investigate the main variant consequences. Some genetic variants in vascular adhesion molecule-1 gene promoter and endothelial nitric oxide synthase gene were associated with higher levels of hemolysis and stroke events. They modify important transcription factor binding sites or disturb the corresponding protein structure/function. Our findings emphasize the relevance of the genetic variants in modulating the degree of hemolysis and development of cerebral vasculopathy due to their effect on gene expression, modification of protein biological activities related with erythrocyte/endothelial interactions and consequent hemorheological abnormalities in SCA.