Browsing by Author "Silva, Carla"
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- Adult-onset form in VLCAD deficiency: seven casesPublication . Sousa, Carmen; Marcão, Ana; Nogueira, Célia; Fonseca, Helena; Rocha, Hugo; Silva, Carla; Guimas, Arlindo; Evangelista, Teresinha; Maré, Rui; Vilarinho, LauraVery long chain acyl-Co-A dehydrogenase deficiency (VLCADD, MIM 201475) is an autosomal recessive disorder characterized by impaired mitochondrial β-oxidation of fatty acids with a chain length between 14 and 18 carbons. The prevalence of VLCAD deficiency in Portugal is 1/101,613. VLCADD has three forms of clinical presentation: severe early-onset; intermediate with childhood onset and adult-onset, of mild severity, characterized by exercise intolerance, myalgia and recurrent episodes of rhabdomyolysis. The development of electrospray ionization tandem mass spectrometry (MS/MS) has allowed beyond the screening of neonatal forms a marked improvement on diagnosis of the adult onset form. The authors report the acyl-carnitines profile that revealed accumulation of tetradecenoyl carnitine (C14:1) in seven individuals with clinical symptoms with the ages between 11 and 63. The eldest patient was diagnosed at the age of 63 years. These results were confirmed by molecular ACADVL gene analysis. When rhabdomyolysis is present in a patient, and after differential diagnosis, it is important to consider the possibility of a VLCAD deficiency. This late-onset form may be undetectable by acyl-carnitine profile in asymptomatic period, and only in crisis is informative. However, if VLCADD is considered the molecular analysis of ACADVL should be performed in all suspected cases.
- Characterization of Mycobacterium tuberculosis resistant strains circulating in a Lisbon hospital: a 2 years studyPublication . Perdigão, João; João, Inês; Silva, Carla; Portugal, Isabel; Jordão, Luísa
- Clonal expansion across the seas as seen through CPLP-TB database: a joint effort in cataloguing Mycobacterium tuberculosis genetic diversity in Portuguese-speaking countriesPublication . Perdigão, João; Silva, Carla; Diniz, Jaciara; Pereira, Catarina; Machado, Diana; Ramos, Jorge; Silva, Hugo; Abilleira, Fernanda; Brum, Clarice; Reis, Ana J.; Macedo, Maíra; Scaini, João L.; Silva, Ana B.; Esteves, Leonardo; Macedo, Rita; Maltez, Fernando; Clemente, Sofia; Coelho, Elizabeth; Viegas, Sofia; Rabna, Paulo; Rodrigues, Amabélia; Taveira, Nuno; Jordao, Luísa; Kritski, Afrânio; Silva, José Lapa e; Mokrousov, Igor; Couvin, David; Rastogi, Nalin; Couto, Isabel; Pain, Arnab; McNerney, Ruth; Clark, Taane G.; von Groll, Andrea; Dalla-Costa, Elis R.; Rossetti, Maria Lúcia; Silva, Pedro E.A. da; Viveiros, Miguel; Portugal, IsabelTuberculosis (TB) remains a major health problem within the Community of Portuguese Language Speaking Countries (CPLP). Despite the marked variation in TB incidence across its member-states and continued human migratory flux between countries, a considerable gap in the knowledge on the Mycobacterium tuberculosis population structure and strain circulation between the countries still exists. To address this, we have assembled and analyzed the largest CPLP M.tuberculosis molecular and drug susceptibility dataset, comprised by a total of 1447 clinical isolates, including 423 multidrug-resistant isolates, from five CPLP countries. The data herein presented reinforces Latin American and Mediterranean (LAM) strains as the hallmark of M. tuberculosis populational structure in the CPLP coupled with country-specific differential prevalence of minor clades. Moreover, using high-resolution typing by 24-loci MIRU-VNTR, six cross-border genetic clusters were detected, thus supporting recent clonal expansion across the Lusophone space. To make this data available to the scientific community and public health authorities we developed CPLP-TB (available at http://cplp-tb.ff.ulisboa.pt), an online database coupled with web-based tools for exploratory data analysis. As a public health tool, it is expected to contribute to improved knowledge on the M. tuberculosis population structure and strain circulation within the CPLP, thus supporting the risk assessment of strain-specific trends.
- Correlation between streptomycin intermediate-level resistance and gidB mutation in an endemic multidrugresistant tuberculosis clusterPublication . Perdigao, joão; Macedo, Rita; Machado, Diana; Silva, Carla; Jordão, Luísa; Couto, Isabel; Viveiros, Miguel; Portugal, IsabelDevelopment of streptomycin-resistance in Mycobacterium tuberculosis is usually associated with mutations in rpsL and rrs genes, although up to 50% of clinical streptomycin-resistant isolates may present no mutation in either of these genes. The situation in Lisbon Health Region is similar, although mutations in rrs geneare only rarely detected. In the present report we investigate the role of gidB gene mutations in streptomycin resistance. We have analyzed 52 streptomycin-resistant and 30 streptomycin-susceptible Mycobacterium tuberculosis clinical isolates by sequencing and endonuclease analysis of the gidB and rpsL genes. All clinical isolates were genotyped by 12-loci MIRU-VNTR. Semiquantitative drug susceptibility testing was also performed to a select set of isolates to assess the resistance levels towards streptomycin. The gidB gene of 18 streptomycin-resistant isolates was sequenced and four missense mutations were found: F12L (1/18), L16R (18/18), A80P (4/18) and S100F (18/18). The remaining isolates were screened by endonuclease analysis for mutations A80P in gidB and K43R in rpsL gene. Overall, mutation A80P in gidB gene was found in 7 streptomycin-resistant isolates and 12 streptomycin-susceptible multidrug resistant isolates. Also noteworthy, comparison of the distribution of gidB, rpsL and rrs mutations revealed that gidB A80P mutation was only present in isolates without rpsL and rrs mutations. Moreover, this specific mutation was found among all isolates belonging to genetic cluster Q1. Streptomycin quantitative drug susceptibility testing showed that isolates carrying the GidB A80P mutation were streptomycin intermediate-level resistant and that standard drug susceptibility testing yielded inconsistent results probably due to borderline resistance. Bioinformatic analysis on the degree of conservation showed that the GidB A80P mutation is predicted to affect protein function. We conclude that gidB mutations may explain the high number of streptomycin-resistant strains with no mutation in rpsL or rrs. These mutations might occasionally confer undetected streptomycin low level resistance in regular drug susceptibility testing. Also, GidB A80P mutations may serve as surrogate markers for Q1 cluster isolates that are associated with multidrug/extensively drug-resistant tuberculosis.
- Deletion analysis on multidrug resistant Mycobacterium tuberculosis in PortugalPublication . Silva, Carla; Perdigão, João; Couto, Isabel; Viveiros, Miguel; Jordão, Luísa; Portugal, IsabelDeletion analysis on multidrug-resistant Mycobacterium tuberculosis in Portugal Carla Silva1,4, João Perdigão1, Isabel Couto2,3, Miguel Viveiros2,5, Luísa Jordão4 and Isabel Portugal1 1URIA, Centro de Patogénese Molecular, Faculdade de Farmácia da Universidade de Lisboa, 2Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), 3Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, 4Departamento de Doenças Infecciosas, Instituto Nacional de Saúde Dr Ricardo Jorge, 5COST ACTION BM0701 (ATENS) Portugal is one of the European countries with the highest incidence of tuberculosis (TB), with a notification rate higher than 20/100 000 inhabitants. One of the biggest threats to TB control is the emergence of infection case with features of multidrug resistance (MDR-TB) and extensive drug-resistance (XDR-TB) TB. Most strains of MDR-TB circulating in the Lisbon area belong to a particular family of genetically related strains, the Lisboa family, detected in the 90’s. The prevalence of this family of strains has increased over the years, and represented more than 85% of the MDR-TB strains in the year of 2008. XDR-TB has been recently derived from MDR-TB strains and account for about 50% of these, the majority belonging to Lisboa family. Lisboa family represent a serious problem regarding TB control in Portugal and its prevalence in recent years suggests that these strains may have selective advantages over others. In an attempt to define the evolutionary origin and the identification of factors that favors the continued existence and spread of Lisboa strains in the Portuguese setting, genomic deletions in seven MDR-TB clinical isolates representative of the principal clusters circulating in Portugal were studied. Previously characterized by 24 loci MIRU-VNTR, the isolates were analyzed for the presence or absence of the genomic regions pks15/1, RD105 and RD174. We verified that five of the 7 isolates analyzed belong to the Euro American Lineage, with the 7bp deletion on pks15/1 gene, with other two belonging to the East-Asia Lineage (including the Beijing family strains), characterized by the presence of an intact pks15/1 gene and absence of RD105 region. Further analysis of the Euro American Lineage strains revealed that four strains, including the two representative strains of Lisboa family, present the same second genomic deletion – RD174, a marker for the sublineage West Africa; the remaining isolate is still under study, and more analysis are ongoing. We conclude that the circulating strains present some lineage diversity, with the predominant strains, Lisboa family, belonging to the West Africa Sublineage. Such, may be indicative of selective advantages and/or better adaptation to the host population in the presence of circulating Beijing strains.
- Dynamics and Development of Extensively Drug-resistant Tuberculosis, PortugalPublication . Perdigao, Joao; Silva, D.; Pereira, V.; Macedo, Rita; Silva, Carla; Machado, Diana; Couto, Isabel; Viveiros, Miguel; Jordão, Luísa; Portugal, IsabelThe development of multidrug-resistant (MDR) and extensive drug-resistant (XDR) tuberculosis(TB) combined with subsequent transmission constitutes a serious threat to the effective control of tuberculosis in several countries. Lisbon Health Region, despite great progresses in TB management still presents a high number of MDR/XDR-TB cases. The development of this type of resistance is the result of adaptative selection of Mycobacterium tuberculosis strains that acquire and accumulate specific mutations at specific genes. The presently known mechanisms of drug resistance include the modification or overexpression of drug targets, inactivation of drug- activator enzymes and overexpression of drug-modifying enzymes. Although the molecular basis of resistance of MDR/XDR-TB strains circulating in Lisbon has already been addressed in different studies, the dynamics or mode of resistance acquisition that have lead to the different circulating strains is still partially unclear. In the present study we have genotyped and screened a set of 44 MDR/XDR-TB isolates for mutations in tlyA, gyrA, rrs and eis genes. We have determined the most prevalent mutations found in each gene to be Ins755GT in tlyA, A1401G in rrs, G-10A in eis and S91P in gyrA. Two genetic clusters previously known to be associated with XDR-TB were detected, Lisboa3 and Q1, containing 27 and 17 isolates, respectively. Lisboa3 strains isolated in the 90’s with the same mutational profile of recent XDR-TB Lisboa3 strains were found, emphasizing the ancient XDR-TB problem in the region. Also investigated was the resistance level conferred by eis G-10A mutations, revealing that eis G-10A mutations may result in an undetectable AMK resistance. We concluded by analyzing the mutational distribution found by genetic cluster that in Q1 cluster two mutations, gyrA D94A and rrs A1401G, were enough to ensure development of XDR-TB from a multidrug resistant strain. Moreover, in Lisboa3 cluster it was possible to determine that the development of kanamycin low-level resistance mediated by eis promoter mutations was at the origin of independent emergence of several XDRTB strains that can be discriminated within Lisboa3 genetic cluster by tlyA mutations.
- Emergence of multidrug-resistant Mycobacterium tuberculosis of the Beijing lineage in Portugal and Guinea-Bissau: a snapshot of moving clones by whole-genome sequencingPublication . Perdigão, João; Silva, Carla; Maltez, Fernando; Machado, Diana; Miranda, Anabela; Couto, Isabel; Rabna, Paulo; Florez de Sessions, Paola; Phelan, Jody; Pain, Arnab; McNerney, Ruth; Hibberd, Martin L.; Mokrousov, Igor; Clark, Taane G.; Viveiros, Miguel; Portugal, IsabelThe Beijing genotype comprises a highly disseminated strain type that is frequently associated with multidrug resistant (MDR) tuberculosis (TB) and increased transmissibility but, countries such as Portugal and Guinea-Bissau fall outside the regions phylogeographically associated with this specific genotype. Nevertheless, recent data shows that this genotype might be gradually emerging in these two countries as an underlying cause of primary MDR-TB. Here, we describe the emergence of Mycobacterium tuberculosis Beijing strains associated with MDR-TB in Portugal and Guinea-Bissau demonstrating the presence of the well described superclusters 100-32 and 94-32 in Portugal and Guinea-Bissau, respectively. Genome-wide analysis and comparison with a global genomic dataset of M. tuberculosis Beijing strains, revealed the presence of two genomic clusters encompassing isolates from Portugal and Guinea-Bissau, GC1 (n = 121) and GC2 (n = 39), both of which bore SNP signatures compatible with the 100-32/B0/W148 and 94-32/Central Asia Outbreak clades, respectively. Moreover, GC2 encompasses a cross-border cluster between Portugal, Guinea-Bissau and Brazil thus supporting migration-associated introduction of MDR-TB and subsequent clonal expansion at the community-level. The comparison with global Beijing datasets demonstrates the global reach of the disease and its complex dissemination across multiple countries while in parallel there are clear microevolutionary trajectories towards extensively drug resistant TB.
- Estudo da imunidade adquirida pela vacinação contra a COVID-19 em profissionais de saúde do INSA: relatório de execuçãoPublication . Guiomar, Raquel; Santos, Ana João; Melo, Aryse; Ramalhete, Sara; Costa, Inês; Henriques, Camila; Matos, Rita; Rodrigues, Ana Paula; Irina, Kislaya; Silva, Anabela Santos; Roque, Carla; Silva, Carla; Aguiar, Joaquim; Dias, Carlos; Machado, Jorge; Graça, Fátima; Graça, António Silva; Machado, AusendaO presente relatório de execução, elaborado pelo Departamento de Epidemiologia, o Departamento de Doenças Infeciosas e os Serviços de Saúde Ocupacional do INSA, apresenta os resultados de implementação dos 12 meses do estudo, desenvolvido para estimar a efetividade da vacina contra a COVID-19 em profissionais de saúde do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA). Neste relatório é apresentada informação sobre a execução do estudo, incluindo descrição dos participantes e da sua participação nos diferentes momentos e etapas, bem como a informação recolhida através das diferentes fontes de dados. O estudo prospetivo de coorte teve como principal objetivo seguir grupos de profissionais vacinados e não-vacinados durante 12 meses para estimar a efetividade da vacina contra a COVID-19 nos trabalhadores do INSA e analisar a resposta imunológica humoral através da deteção de anticorpos vacinais. O estudo iniciou-se em janeiro de 2021 e terminou a 16 fevereiro 2022.
- Exploring a link between mycobacteria structure and virulencePublication . Silva, Carla; Perdigao, Joao; Alverca, Elsa; Matos, António Pedro Alves; Carvalho, Patrícia A; Portugal, Isabel; Jordão, LuísaTuberculosis (TB) is a major health problem. The emergence of multidrug resistant (MDR)Mycobacterium tuberculosis (Mtb) isolates confounds treatment strategies. In Portugal, cases of MDR-TB are reported annually with increased incidence noted in Lisbon. The majority of these MDR-TB cases are due to closely-related mycobacteria known collectively as Lisboa family and Q1 cluster. The genetic determinants linked to drug resistance have been exhaustively studied resulting in the identification of family and cluster specific mutations. Nevertheless, little is known about other factors involved in drug resistance development. Here we focused on the study of morphological and structural features of Mtb isolates, collected during 2008-2009 in Lisbon, in order to complement the genetic analysis. For this propose scanning and transmission electron microscopy techniques were used. Particular attention was given to Lisboa family and Q1 cluster isolates since together they account for the majority of reported MDR-TB cases. This analysis allowed the identification of structural differences, such as cell envelope thickness, between Mtb clinical isolates, which are correlated with antibiotic resistance. The infection of human monocyte derived macrophages allowed us to document the relative selective advantage of Lisboa family isolates over other circulating Mtb isolates.
- Exploring the contribution of mycobacteria characteristics in the interaction with human macrophagesPublication . Silva, Carla; Perdigao, Joao; Alverca, Elsa; de Matos, António Pedro Alves; Carvalho, Patrícia A; Portugal, Isabel; Jordão, LuísaTuberculosis (TB) is a major health problem. The emergence of multidrug resistant (MDR) Mycobacterium tuberculosis (Mtb) isolates confounds treatment strategies. In Portugal, cases of MDR-TB are reported annually with increased incidence noted in Lisbon. The majority of these MDR-TB cases are due to closely-related mycobacteria known collectively as Lisboa family and Q1 cluster. The genetic determinants linked to drug resistance have been exhaustively studied resulting in the identification of family and cluster specific mutations. Nevertheless little is known about other factors involved in drug resistance development. Here, for the first time, we complemented the genetic analysis with the study of morphological and structural features of Lisboa family and Q1 cluster isolates by using scanning and transmission electron microscopy. This analysis allowed the identification of structural differences, such as cell envelope thickness, between Mtb clinical isolates, which are correlated with antibiotic resistance. The infection of human monocyte derived macrophages allowed us to document the relative selective advantage of Lisboa family isolates over other circulating Mtb isolates.
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