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Improved nucleic acid testing strategies to detect and discriminate veterinary relevant Mycobacterium tuberculosis complex members
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Os membros do complexo Mycobacterium tuberculosis (MTC) são agentes causadores de tuberculose em humanos e animais. A tuberculose bovina tem sido sujeita nas últimas décadas a programas de erradicação bastante dispendiosos, na maioria dos países desenvolvidos, envolvendo a análise laboratorial de tecidos de animais suspeitos para a detecção dos membros do MTC, nomeadamente Mycobacterium bovis. O diagnóstico definitivo é obtido através da cultura bacteriológica, o que pode levar 6-12 semanas, período durante o qual a carcaça do animal suspeito e a exploração de origem permanecem sob embargo sanitário. Neste trabalho, descreve-se um protocolo de extracção de DNA de fácil utilização adaptado aos tecidos, o qual é acoplado a um semi-nested PCR em tempo real, utilizando como alvo a IS6110, por forma a melhorar a detecção directa de bactérias pertencentes ao MTC em animais, abreviando o período necessário ao diagnóstico. O ensaio foi avaliado num grupo de 128 amostras de tecido provenientes de bovinos, javalis, veados e raposas. O desempenho global do teste corresponde a uma sensibilidade e especificidade de diagnóstico de 98,2% e 88,7%, respectivamente. Foi observado um coeficiente kappa de 0,859 entre o ensaio de semi-nested PCR e a cultura bacteriológica. Este ensaio permite a detecção rápida de micobactérias tuberculosas em amostras de animais com alta sensibilidade e especificidade, sendo acessível e de baixo custo para uma utilização num laboratório de diagnóstico veterinário.
As espécies do MTC são geneticamente muito semelhantes, mas podem divergir na sua epidemiologia, nomeadamente na distribuição geográfica e preferência pelo hospedeiro, factores de virulência e padrões de susceptibilidade antimicrobiana. No entanto, o diagnóstico laboratorial convencional não diferencia rotineiramente as espécies do MTC. Foi desenvolvido um algoritmo de identificação rápido e robusto, baseado em PCR em tempo real, dirigido para cinco alvos genómicos para a identificação das espécies do MTC vulgarmente associadas à tuberculose nos bovinos e outros animais. O primeiro passo permite a confirmação dos membros do MTC nas culturas, através da detecção da IS6110, ou como uma espécie micobacteriana, pela presença do 16S rDNA. Se uma espécie do MTC for identificada, o segundo passo do algoritmo permite avaliar a presença ou ausência das regiões genómicas RD1, RD4 e RD9. O padrão correspondente permite inferir a espécie do isolado como M. tuberculosis (se todas as RDs estiverem presentes), M. caprae (se apenas a RD1 e RD4 estiverem presentes) ou M. bovis (se apenas a RD1 estiver presente). O algoritmo de identificação desenvolvido demonstrou um coeficiente kappa de 0,970 com o resultado da análise bacteriológica. O ensaio pode ser implementado em laboratórios de diagnóstico veterinário, especialmente em laboratórios de referência.
Tem-se registado uma procura crescente por métodos de diagnóstico de doenças infecciosas rápidos, de fácil utilização e acessíveis, passíveis de serem utilizados em pontos-de-decisão. A detecção dos membros do MTC é geralmente realizada por diversos métodos convencionais baseados na cultura, que normalmente necessitam de oito semanas. Foram também desenvolvidas estratégias de diagnóstico molecular, mas a maioria requer operadores qualificados e equipamentos e infra-estruturas sofisticadas. Recentemente, a técnica de Loop-Mediated Isothermal Amplification (LAMP) mostrou-se promissora para o desenvolvimento de testes rápidos, de baixo custo, sensíveis e específicos para a detecção de agentes patogénicos. Neste trabalho, foram optimizados dois sistemas LAMP em formato duplex (dLAMP) para a identificação do MTC e Mycobacterium tuberculosis, e do MTC e M. bovis, apresentando valores de sensibilidade e especificidade comparáveis a outras abordagens em que se utiliza o PCR convencional. Os resultados das amplificações são avaliados colorimetricamente utilizando dispositivos de fluxo lateral, simples e comercialmente disponíveis, para a detecção de ácidos nucleicos (NALF).
Os resultados apresentados nesta dissertação contribuem para a melhoria das estratégias de diagnóstico molecular existentes no combate à tuberculose animal.
Members ofMycobacterium tuberculosiscomplex (MTC)are causative agents of tuberculosis in both humans and animals.Bovine tuberculosis has been tackled for decades by costly eradication programs in most developed countries, involving thelaboratorial testing of tissue samples from allegedly infected animals for detection of MTCmembers, namely Mycobacterium bovis. Definitive diagnosis is usually achieved by bacteriological culture, which may take up to 6-12 weeks, during which the suspectanimal carcass and herd are under sanitary arrest.In this work we describea user-friendly DNA extraction protocol adapted for tissues andcoupled with an IS6110-targeted semi-nested real-time PCR assay to enhance the direct detection of MTC bacteria in animal specimens, reducing the time to achieve a diagnosis. The assay was evaluated with a group of 128 freshtissue specimens collected from bovines, wild boars, deer and foxes. Overall, the full test performance corresponds to a diagnostic sensitivity and specificity of 98.2% and 88.7%, respectively. An observed kappa coefficient was estimated in 0.859 for the overall agreement between the semi-nested PCR assay and the bacteriological culture. This novel IS6110-targeted assay allows the fast detection of tuberculous mycobacteria in animal specimens with very high sensitivity and specificity, beingamenable and cost effective for use in the routine veterinary diagnostic laboratory with further automation possibilities.MTC species are genetically very similar but may differ in their epidemiology, namely geographic distributionand host preferences, virulence traits and antimicrobial susceptibility patterns. However, the conventional laboratory diagnosis does not routinely differentiate between the species ofthe MTC. We developedarapid and robust two-step five-target probe-based real-time PCR identification algorithm, based on genomic deletion analysis, to identify the MTC species most commonly associated with TB in livestock and other animals.The first step allows the confirmation of the cultures as MTC members, by targeting their IS6110element, or as a mycobacterial species, if only a 16S rDNA product is detected in the duplex amplification reaction. If a MTC member is identified, the second amplification step allows to assess the presence or absence of the RD1, RD4 and RD9 genomic regions. The correspondent pattern allows to infer the species of the isolate as M. tuberculosis(if all RDs are present), M. caprae(if only RD1 and RD4 are present) and M. bovis(if only RD1 is present). The identification algorithmdevelopedpresented an almost perfect agreement with the results of the routine bacteriological analysis, with a kappa coefficient of 0.970.The assay is able to be adaptable to automation and implementation in the routine diagnostics framework of veterinary diagnostics laboratories, with a particular focus for reference laboratories. Rapid, user-friendly and affordable diagnostictests for use in the point-of-decision or point-of-care settings are in high demand globally. Detection of MTC members is generally performedby cumbersome conventional culture-based methods,whichusually takesup to eight weeks.Molecular diagnosis strategies were also developed but most of these requires skilled operators and sophisticatedequipmentsand facilities. More recently, the Loop-Mediated Isothermal Amplification (LAMP) technique showed promise for the development of rapid, low-cost, sensitive and specific testsfor detecting pathogens.In this work we have optimized duplex LAMP (dLAMP) assays for the identification of MTC and Mycobacterium tuberculosis, and MTCand M. bovis, presenting similar sensitivities and specificitieswhen compared to standard PCRapproaches. The amplification results are assessed colorimetricallyby using simple and commercially available nucleic acid lateral flow (NALF) strips.With the work described in this dissertation we aim to modestly contributeto the improvement of the existing molecular diagnosis strategiesto combat animal tuberculosis.
Members ofMycobacterium tuberculosiscomplex (MTC)are causative agents of tuberculosis in both humans and animals.Bovine tuberculosis has been tackled for decades by costly eradication programs in most developed countries, involving thelaboratorial testing of tissue samples from allegedly infected animals for detection of MTCmembers, namely Mycobacterium bovis. Definitive diagnosis is usually achieved by bacteriological culture, which may take up to 6-12 weeks, during which the suspectanimal carcass and herd are under sanitary arrest.In this work we describea user-friendly DNA extraction protocol adapted for tissues andcoupled with an IS6110-targeted semi-nested real-time PCR assay to enhance the direct detection of MTC bacteria in animal specimens, reducing the time to achieve a diagnosis. The assay was evaluated with a group of 128 freshtissue specimens collected from bovines, wild boars, deer and foxes. Overall, the full test performance corresponds to a diagnostic sensitivity and specificity of 98.2% and 88.7%, respectively. An observed kappa coefficient was estimated in 0.859 for the overall agreement between the semi-nested PCR assay and the bacteriological culture. This novel IS6110-targeted assay allows the fast detection of tuberculous mycobacteria in animal specimens with very high sensitivity and specificity, beingamenable and cost effective for use in the routine veterinary diagnostic laboratory with further automation possibilities.MTC species are genetically very similar but may differ in their epidemiology, namely geographic distributionand host preferences, virulence traits and antimicrobial susceptibility patterns. However, the conventional laboratory diagnosis does not routinely differentiate between the species ofthe MTC. We developedarapid and robust two-step five-target probe-based real-time PCR identification algorithm, based on genomic deletion analysis, to identify the MTC species most commonly associated with TB in livestock and other animals.The first step allows the confirmation of the cultures as MTC members, by targeting their IS6110element, or as a mycobacterial species, if only a 16S rDNA product is detected in the duplex amplification reaction. If a MTC member is identified, the second amplification step allows to assess the presence or absence of the RD1, RD4 and RD9 genomic regions. The correspondent pattern allows to infer the species of the isolate as M. tuberculosis(if all RDs are present), M. caprae(if only RD1 and RD4 are present) and M. bovis(if only RD1 is present). The identification algorithmdevelopedpresented an almost perfect agreement with the results of the routine bacteriological analysis, with a kappa coefficient of 0.970.The assay is able to be adaptable to automation and implementation in the routine diagnostics framework of veterinary diagnostics laboratories, with a particular focus for reference laboratories. Rapid, user-friendly and affordable diagnostictests for use in the point-of-decision or point-of-care settings are in high demand globally. Detection of MTC members is generally performedby cumbersome conventional culture-based methods,whichusually takesup to eight weeks.Molecular diagnosis strategies were also developed but most of these requires skilled operators and sophisticatedequipmentsand facilities. More recently, the Loop-Mediated Isothermal Amplification (LAMP) technique showed promise for the development of rapid, low-cost, sensitive and specific testsfor detecting pathogens.In this work we have optimized duplex LAMP (dLAMP) assays for the identification of MTC and Mycobacterium tuberculosis, and MTCand M. bovis, presenting similar sensitivities and specificitieswhen compared to standard PCRapproaches. The amplification results are assessed colorimetricallyby using simple and commercially available nucleic acid lateral flow (NALF) strips.With the work described in this dissertation we aim to modestly contributeto the improvement of the existing molecular diagnosis strategiesto combat animal tuberculosis.
Descrição
Palavras-chave
Microbiologia médica Tuberculose bovina Diagnóstico Mycobacterium tuberculosis
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical