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Universidade Do Estado Do Rio De Janeiro Centro Biomédico Faculdade De Ciências Médicas Edgard De Freitas Vianna Identifica

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Universidade Do Estado Do Rio De Janeiro Centro Biomédico Faculdade De Ciências Médicas Edgard De Freitas Vianna Identifica Universidade do Estado do Rio de Janeiro Centro Biomédico Faculdade de Ciências Médicas Edgard de Freitas Vianna Identificação das espécies do Complexo Burkholderia cepacia em pacientes com fibrose cística: comparação entre as técnicas de Matrix-Assisted Laser Desorption-Ionization - Time of Flight Mass Spectrometry e o sequenciamento do gene recA Rio de Janeiro 2017 Edgard de Freitas Vianna Identificação das espécies do Complexo Burkholderia cepacia em pacientes com fibrose cística: comparação entre as técnicas de Matrix-Assisted Laser Desorption-Ionization - Time of Flight Mass Spectrometry e o sequenciamento do gene recA Dissertação apresentada, como requisito parcial para obtenção do título de Mestre, ao programa de Pós- graduação em Microbiologia, da Universidade do Estado do Rio de Janeiro. Área de concentração: Microbiologia Médica Humana. Orientadora: Prof.a Dra. Elizabeth de Andrade Marques Rio de Janeiro 2017 UERJ/REDE SIRIUS/BIBLIOTECA CB-A CATALOGAÇÃO NA FONTE V617 Vianna, Edgard de Freitas. Identificação das espécies do Complexo Burkholderia cepacia em pacientes com fibrose cística: comparação entre as técnicas de Matrix- Assisted Laser Desorption-Ionization - Time of Flight Mass Spectrometry e o sequenciamento do gene recA. / Edgard de Freitas Vianna. – 2017. 81 f. Orientadora: Elizabeth de Andrade Marques. Dissertação (Mestrado) – Universidade do Estado do Rio de Janeiro, Faculdade de Ciências Médicas. Pós-graduação em Microbiologia. 1. Complexo Burkholderia cepacia. 2. Fibrose Cística. 3. Matrix Assisted Laser Desorption-Ionization - Teses. I. Marques, Elizabeth de Andrade. II. Universidade do Estado do Rio de Janeiro. Faculdade de Ciências III. Título. Médicas. CDU 616-008.87 Autorizo, apenas para fins acadêmicos e científicos, a reprodução total ou parcial desta dissertação, desde que citada a fonte. _________________________ _________________________ Assinatura Data Edgard de Freitas Vianna Identificação das espécies do Complexo Burkholderia cepacia em pacientes com fibrose cística: comparação entre as técnicas de Matrix-Assisted Laser Desorption-Ionization - Time of Flight Mass Spectrometry e o sequenciamento do gene recA Dissertação apresentada, como requisito parcial para obtenção do título de Mestre, ao programa de Pós- graduação em Microbiologia, da Universidade do Estado do Rio de Janeiro. Área de concentração: Microbiologia Médica Humana. Aprovada em 30 de março de 2017. Orientadora: Prof.a Dra. Elizabeth de Andrade Marques Faculdade de Ciências Médicas - UERJ Banca Examinadora: Prof.ª Dra. Mara Lucia Penna Queiroz Faculdade de Ciências Médicas - UERJ Prof.ª Dra.Vania Lucia Carreira Merquior Faculdade de Ciências Médicas – UERJ Prof.ª Dra. Mônica de Cassia Firmida Hospital Universitário Pedro Ernesto - UERJ Rio de Janeiro 2017 DEDICATÓRIA À Deus, por mostrar que é Ele quem efetua tanto o querer quanto o realizar, de acordo com a boa vontade dEle e que seus pensamentos são maiores que os meus. AGRADECIMENTOS A minha esposa Maristela, por ser mais que sonhava, meu amor, minha companheira, minha amiga, por possibilitar a construção deste sonho, entender e acreditar em mim em todos os momentos. A minha família, por toda a dedicação depositada em mim, fazendo-me alcançar voos mais altos e por entender a minha ausência. A minha orientadora Prof.ª Dra. Elizabeth Marques, por me conceder a honra de ser seu aluno, um exemplo de dedicação e profissionalismo, um exemplo de vida. Obrigado por todo ensinamento transmitido ao longo dessa jornada. Ao professor Robson Leão e aos meus amigos do Laboratório 2, Meyvianne, Mila, Leonardo, Márcia, Géssica, Daniele Lima, Daniele Pacheco, Daiana, pela atenção, amizade e por todo ensinamento transmitido. A minha “equipe CBc”, Cassiana, Flávia, Jéssica, Elisa, Anne e Érica, por todo companheirismo, ensinamento e ajuda nos momentos que precisei. Aos amigos da minha turma de mestrado, Fabiane, Ohana, Deise, Luciana e Bruno pelo prazer de conhecê-los. Aos meus Colegas e amigos do Hospital Rios D’or, Laboratório Richet e do Genoma- UERJ pelo incentivo durante todo o projeto. Aos professores do Departamento de Microbiologia, Imunologia e Parasitologia da UERJ que contribuíram para minha formação profissional e pessoal. A toda a equipe técnica da Disciplina de Microbiologia e Imunologia, em especial aos técnicos Rose e Denilson. À secretária Carla, pela atenção, dedicação e amor com que conduz seu trabalho e nos auxilia nas questões burocráticas. A todos aqueles que torceram por mim e que contribuíram direta ou indiretamente para que este trabalho alcançasse seus objetivos. Muito obrigado! A personalidade humana é uma construção sofisticada, cedo ou tarde não sobra pedra sobre pedra. Recolher nossos fragmentos e nos reinventar é o que nos diferencia. Augusto Cury RESUMO VIANNA, Edgard de Freitas. Identificação das espécies do Complexo Burkholderia cepacia em pacientes com fibrose cística: comparação entre as técnicas de Matrix-Assisted Laser Desorption-Ionization - Time of Flight Mass Spectrometry e o sequenciamento do gene recA. 2017. 81 f. Dissertação (Mestrado em Microbiologia) – Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2017. O Complexo Burkholderia cepacia (CBc) compreende 20 espécies intimamente relacionadas que se destacam como patógenos oportunistas na Fibrose Cística (FC) e estão associadas a um impacto considerável na morbimortalidade desses pacientes. As espécies e subtipos filogenéticos dentro da espécie Burkholderia cenocepacia variam entre si em relação a transmissibilidade e a virulência. Devido a inconsistências na correta identificação das espécies do CBc por métodos fenotípicos, os métodos moleculares são recomendados. Entretanto não existe uma metodologia molecular universal que possa ser aplicada para todos os micro-organismos, especialmente em laboratórios clínicos. Nesse sentido, a metodologia baseada em espectrometria de massa surge como uma alternativa. O objetivo deste estudo foi avaliar o desempenho do Matrix Associated Laser Desorption-Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS) comparado à identificação das espécies do CBc através do sequenciamento do gene recA em pacientes com FC. Foram analisadas 43 amostras obtidas de diferentes espécimes clínicos no período de janeiro de 2014 a fevereiro de 2016. Um total de 18 (41,86%) amostras de Burkholderia vietnamiensis, 14 (32,55%) Burkholderia cenocepacia subtipo IIIB, oito (18,6%) Burkholderia cenocepacia subtipo IIIA, duas (4,65%) Burkholderia contaminans e uma (2,32%) Burkholderia cepacia foram identificadas pelo sequenciamento do gene recA. A identificação pelo MALDI-TOF MS foi avaliada considerando o cultivo prévio em Ágar Sangue de Carneiro (ASC) e Columbia Ágar Base (CAB). 81,39% das amostras provenientes do ASC foram identificadas a nível de gênero (log ≥1.70-1.99) e 41,86% a nível de espécie (log ≥2.00), correspondendo a 11 amostras de B. vietnamiensis (61,11%), quatro de B. cenocepacia IIIB (28,57%) e duas de B. cenocepacia IIIA (25%). Apenas 39,53% das amostras cultivadas em CAB foram identificadas a nível de gênero e 16,27% em espécie: B. vietnamiensis (n=4; 22,22%) e B. cenocepacia IIIB (n=2; 14,28%). A amostra de B. cepacia foi identificada a partir de ambos os meios e as duas amostras de B. contaminans não foram identificadas. O desempenho do MALDI- TOF MS variou com a utilização de diferentes meios de cultura e a identificação a nível de espécie foi inferior a 50% considerando os pontos de corte estabelecidos pelo fabricante. Assim, avaliamos se a redução dos pontos de corte aumentaria a acurácia da identificação. Com a minoração do escore para ≥1.70-1.99 (considerado como identificação confiável a nível de gênero), foram identificadas mais três amostras de B. vietnamiensis, quatro de B. cenocepacia IIIA e sete de B. cenocepacia IIIB, correspondendo a um aumento de 32,55% (em relação ao escore ≥ 2.00). Entretanto o número de amostras analisadas não permite que esse critério seja utilizado sendo necessário estudos adicionais. Apesar da limitação do MALDI-TOF MS como método para tipagem, a utilização de ferramentas de bioinformática para análise dos espectros de massa obtidos no MALDI-TOF MS permitiu o agrupamento de 96% dos subtipos filogenéticos IIIA e IIIB de cepas B. cenocepacia. Observamos também através dos nós formados na árvore filogenética a proximidade das amostras relacionadas ao mesmo paciente, porém a ausência de comparação com um método de tipagem de referência não permite inferir a sua utilização para fins epidemiológicos. Palavras-chave: Complexo Burkholderia cepacia. Fibrose Cística. Matrix Associated Laser Desorption-Ionization - Time of Flight Mass Spectrometry. Sequenciamento do gene recA. ABSTRACT VIANNA, Edgard de Freitas. Identification of Burkholderia cepacia complex species isolated from patients with cystic fibrosis: performance ofMatrix-Assisted Laser Desorption-Ionization - Time of Flight Mass Spectrometry compared to recA sequencing. 2017. 81 f. Dissertação (Mestrado em Microbiologia) – Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2017. Burkholderia cepacia Complex (CBc) comprises 20 closely related species that stand out as opportunistic pathogens in Cystic Fibrosis (CF) and are associated with a considerable impact on the morbimortality of these patients. Phylogenetic species and subtypes within the Burkholderia cenocepacia species vary in relation to transmissibility and virulence. Due to inconsistencies in the correct identification of CBc species
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