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Caracterização genotípica de isolados de Mycobacterium tuberculosis em Lisboa e desenvolvimento de um método rápido de deteção baseado em PCR para estirpes Lisboa3 E Q1
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Resumo(s)
A tuberculose (TB) persiste como um importante desafio ao nível de saúde pública e
continua a reunir esforços e atenções no que toca à sua monitorização e evolução
epidemiológica apesar de presentemente constituir um risco menor neste domínio.
Contudo a TB multirresistente (MDR-TB) suscita ainda muita inquietação pela acrescida
resistência a antibióticos e facilidade de disseminação, e no caso concreto de Portugal,
mesmo tendo em conta a melhoria da situação epidemiológica ao longo das últimas três
décadas, a MDR-TB carece de dados concretos e de estratégias delineadas para o combate
da mesma.
Neste trabalho pretendeu-se desenvolver um método rápido de caracterização genotípica
para estirpes de reconhecido interesse epidemiológico e caracterizar do ponto de vista da
epidemiologia molecular um conjunto de isolados clínicos provenientes de unidades de
saúde da Região de Saúde de Lisboa. A região de Lisboa, a par da região do Porto, é uma
região que ainda é afetada de forma significativa pela TB, com os últimos dados a
apontarem para uma taxa de notificação de 20,3 casos por 100 000 habitantes. Estudos
anteriores revelam também a existência de duas estirpes multirresistentes em circulação
nesta região e com reconhecida importância epidemiológica: Lisboa3 e Q1.
O presente estudo compreendeu duas fases, em que numa primeira fase se procurou
otimizar e validar um método de caracterização genotípica rápida baseada em ARMS PCR (Amplification-Refratory Mutation System PCR) e PCR multiplex com
amplificação dirigida a uma sub-região da IS6110 (característica do complexo
Mycobacterium tuberculosis), ao polimorfismo fbpc103 específico da sub-linhagem LAM
(da qual fazem parte as estirpes Q1 e Lisboa3) e, para os SNP´s específicos das estirpes
Lisboa3 e Q1. A segunda fase do estudo, executada após a validação do método, baseou se na descrição e caracterização genotípica de 28 isolados provenientes de unidades de
saúde em Lisboa durante os últimos oito anos. Os resultados obtidos mostraram a
existência de nove isolados pertencentes à família LAM, sendo um isolado pertencente à
família Q1 e um outro isolado pertencente à família Lisboa3.
Como método complementar ao desenvolvimento do PCR Multiplex, a técnica MIRU VNTR foi usada para a genotipagem e caracterização da clonalidade dos isolados
estudados. Foi detetado um cluster dentro do conjunto de isolados analisados não
associado a estirpes Lisboa3 nem Q1. O perfil alélico obtido para o isolado positivo para
Lisboa3 pela metodologia PCR Multiplex é extremamente semelhante ao perfil alélico
característico de estirpes Lisboa3, e três dos isolados analisados apresentam outros perfis
alélicos com forte grau de associação a outras linhagens com cada vez mais
preponderância no contexto europeu, como é o caso de Haarlem, S e Beijing.
Tuberculosis (TB) persists as an important public health challenge and continues to gather efforts and attention in terms of its monitoring and epidemiological evolution, despite currently representing a lower risk in this area. However, multidrug-resistant TB (MDR TB) still raises a lot of concern due to increased resistance to antibiotics and ease of dissemination, and in the specific case of Portugal, even taking into account the improvement in the epidemiological situation over the last three decades, MDR-TB lacks concrete data and strategies designed to combat it. The aim of this work was to develop a rapid method of genotypic characterization for strains of recognized epidemiological interest and to characterize, from the point of view of molecular epidemiology, a set of clinical isolates from health units in the Lisbon Health Region. The Lisbon region, along with the Porto region, is a region that is still significantly affected by TB, with the latest data pointing to a notification rate of 20.3 cases per 100,000 inhabitants. Previous studies also reveal the existence of two multidrug resistant strains circulating in this region with recognized epidemiological importance: Lisboa3 and Q1. The present study comprised two phases, in which, in the first phase, an attempt was made to optimize and validate a rapid genotypic characterization method based on ARMS-PCR (Amplification-Refractory Mutation System PCR) and multiplex PCR with amplification directed at a sub-region of IS6110 (characteristic of the Mycobacterium tuberculosis complex), while fbpc103 polymorphism specific to the LAM sub-lineage (of which the Q1 and Lisboa3 strains are part) and, for the specific SNPs of the Lisboa3 and Q1 strains. The second phase of the study, carried out after validation of the method, was based on the description and genotypic characterization of 28 isolates from health units in Lisbon over the last eight years. The results obtained showed the existence of nine isolates belonging to the LAM family, with one isolate belonging to the Q1 family and another isolate belonging to the Lisboa3 family. As a complementary method to the development of Multiplex PCR, the MIRU-VNTR technique was used for genotyping and characterizing the clonality of the studied isolates. A cluster was detected within the set of analysed isolates not associated with Lisboa3 or Q1 strains. The allelic profile obtained for the isolate positive for Lisboa3 by PCR Multiplex methodology is extremely similar to the allelic profile characteristic of Lisboa3 strains, and three of the analysed isolates have other allelic profiles with a strong degree of association with other strains with increasing preponderance in the European context, as is the case in Haarlem, S and Beijing.
Tuberculosis (TB) persists as an important public health challenge and continues to gather efforts and attention in terms of its monitoring and epidemiological evolution, despite currently representing a lower risk in this area. However, multidrug-resistant TB (MDR TB) still raises a lot of concern due to increased resistance to antibiotics and ease of dissemination, and in the specific case of Portugal, even taking into account the improvement in the epidemiological situation over the last three decades, MDR-TB lacks concrete data and strategies designed to combat it. The aim of this work was to develop a rapid method of genotypic characterization for strains of recognized epidemiological interest and to characterize, from the point of view of molecular epidemiology, a set of clinical isolates from health units in the Lisbon Health Region. The Lisbon region, along with the Porto region, is a region that is still significantly affected by TB, with the latest data pointing to a notification rate of 20.3 cases per 100,000 inhabitants. Previous studies also reveal the existence of two multidrug resistant strains circulating in this region with recognized epidemiological importance: Lisboa3 and Q1. The present study comprised two phases, in which, in the first phase, an attempt was made to optimize and validate a rapid genotypic characterization method based on ARMS-PCR (Amplification-Refractory Mutation System PCR) and multiplex PCR with amplification directed at a sub-region of IS6110 (characteristic of the Mycobacterium tuberculosis complex), while fbpc103 polymorphism specific to the LAM sub-lineage (of which the Q1 and Lisboa3 strains are part) and, for the specific SNPs of the Lisboa3 and Q1 strains. The second phase of the study, carried out after validation of the method, was based on the description and genotypic characterization of 28 isolates from health units in Lisbon over the last eight years. The results obtained showed the existence of nine isolates belonging to the LAM family, with one isolate belonging to the Q1 family and another isolate belonging to the Lisboa3 family. As a complementary method to the development of Multiplex PCR, the MIRU-VNTR technique was used for genotyping and characterizing the clonality of the studied isolates. A cluster was detected within the set of analysed isolates not associated with Lisboa3 or Q1 strains. The allelic profile obtained for the isolate positive for Lisboa3 by PCR Multiplex methodology is extremely similar to the allelic profile characteristic of Lisboa3 strains, and three of the analysed isolates have other allelic profiles with a strong degree of association with other strains with increasing preponderance in the European context, as is the case in Haarlem, S and Beijing.
Descrição
Palavras-chave
Microbiologia médica Tuberculose Saúde pública Resistência a antibióticos PCR Lisboa3 MDR MIRU-VNTR Q1 Portugal Biologia molecular