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
Ano de defesa: | 2017 |
---|---|
Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | , , |
Tipo de documento: | Dissertação |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade do Estado do Rio de Janeiro
|
Programa de Pós-Graduação: |
Programa de Pós-Graduação em Microbiologia
|
Departamento: |
Centro Biomédico::Faculdade de Ciências Médicas
|
País: |
BR
|
Palavras-chave em Português: | |
Palavras-chave em Inglês: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://www.bdtd.uerj.br/handle/1/14450 |
Resumo: | 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 by phenotypic methods, molecular methods are recommended. However, there is no universal molecular methodology that can be applied to all microorganisms, especially in clinical laboratories. In this sense, the methodology based on mass spectrometry appears as an alternative. The aim of this study was to evaluate the performance of the Matrix Associated Laser Desorption-Ionization (MALDI-TOF MS) compared to Bcc species identification by recA gene sequencing in CF patients. We analyzed 43 samples obtained from different clinical specimens from January 2014 to February 2016. A total of 18 (41.86%) samples of Burkholderia vietnamiensis, 14 (32.55%) Burkholderia cenocepacia subtype IIIB, eight (18.6%) Burkholderia cenocepacia subtype IIIA, two (4.65%) Burkholderia contaminans and one (2.32%) Burkholderia cepacia were identified by sequencing the recA gene. Identification by MALDI-TOF MS was evaluated considering prior culture in Sheep Blood Agar (SBA) and Columbia Agar Base (CAB). 81.39% of the SBA samples were identified at the genus level (log ≥1.70-1.99) and 41.86% at the species level (log ≥2.00), corresponding to 11 samples of B. vietnamiensis (61.11%), four of B. cenocepacia IIIB (28,57%) and two of B. cenocepacia IIIA (25%). Only 39.53% of the samples cultured in CAB were identified at the genus level and 16.27% in species: B. vietnamiensis (n = 4, 22.22%) and B. cenocepacia IIIB (n = 2; 14,28%). B. cepacia sample was identified from both media and the two B. contaminans samples were not identified. Performance of MALDI-TOF MS varied with the use of different culture media and the identification at the species level was less than 50% considering the cut-off points established by the manufacturer. Thus, we evaluated whether the reduction of cut-off points would increase the accuracy of the identification. With the reduction of the escore to ≥1.70-1.99 (considered as reliable identification at genus level), another three samples of B. vietnamiensis, four of B. cenocepacia IIIA and seven of B. cenocepacia IIIB were identified, corresponding to an increase of 32.55% (in relation to the escore ≥ 2.00). However, the number of samples analyzed does not allow this criterion to be used and additional studies are required. Despite the limitations of MALDI-TOF MS as a typing method, the use of bioinformatics tools to analyze the mass spectra obtained in MALDI-TOF MS allowed the clustering of 96% of the phylogenetic subtypes IIIA and IIIB of B. cenocepacia strains. We also observed the proximity of samples related to the same patient through the nodes formed in the phylogenetic tree, but the absence of comparison with a reference typing method does not allow to infer their use for epidemiological purposes. |
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Marques, Elizabeth de Andradehttp://lattes.cnpq.br/5959485578597640Queiroz, Mara Lucia Pennahttp://lattes.cnpq.br/1980792659825205Merquior, Vania Lucia Carreirahttp://lattes.cnpq.br/0742408281665373Fírmida, Mônica de Cássiahttp://lattes.cnpq.br/6507048199597970http://lattes.cnpq.br/0787358470518689Vianna, Edgard de Freitas2021-01-07T15:16:53Z2018-01-312017-03-30VIANNA, 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. 82 f. Dissertação (Mestrado em Microbiologia Médica Humana) - Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2017.http://www.bdtd.uerj.br/handle/1/14450Burkholderia 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 by phenotypic methods, molecular methods are recommended. However, there is no universal molecular methodology that can be applied to all microorganisms, especially in clinical laboratories. In this sense, the methodology based on mass spectrometry appears as an alternative. The aim of this study was to evaluate the performance of the Matrix Associated Laser Desorption-Ionization (MALDI-TOF MS) compared to Bcc species identification by recA gene sequencing in CF patients. We analyzed 43 samples obtained from different clinical specimens from January 2014 to February 2016. A total of 18 (41.86%) samples of Burkholderia vietnamiensis, 14 (32.55%) Burkholderia cenocepacia subtype IIIB, eight (18.6%) Burkholderia cenocepacia subtype IIIA, two (4.65%) Burkholderia contaminans and one (2.32%) Burkholderia cepacia were identified by sequencing the recA gene. Identification by MALDI-TOF MS was evaluated considering prior culture in Sheep Blood Agar (SBA) and Columbia Agar Base (CAB). 81.39% of the SBA samples were identified at the genus level (log ≥1.70-1.99) and 41.86% at the species level (log ≥2.00), corresponding to 11 samples of B. vietnamiensis (61.11%), four of B. cenocepacia IIIB (28,57%) and two of B. cenocepacia IIIA (25%). Only 39.53% of the samples cultured in CAB were identified at the genus level and 16.27% in species: B. vietnamiensis (n = 4, 22.22%) and B. cenocepacia IIIB (n = 2; 14,28%). B. cepacia sample was identified from both media and the two B. contaminans samples were not identified. Performance of MALDI-TOF MS varied with the use of different culture media and the identification at the species level was less than 50% considering the cut-off points established by the manufacturer. Thus, we evaluated whether the reduction of cut-off points would increase the accuracy of the identification. With the reduction of the escore to ≥1.70-1.99 (considered as reliable identification at genus level), another three samples of B. vietnamiensis, four of B. cenocepacia IIIA and seven of B. cenocepacia IIIB were identified, corresponding to an increase of 32.55% (in relation to the escore ≥ 2.00). However, the number of samples analyzed does not allow this criterion to be used and additional studies are required. Despite the limitations of MALDI-TOF MS as a typing method, the use of bioinformatics tools to analyze the mass spectra obtained in MALDI-TOF MS allowed the clustering of 96% of the phylogenetic subtypes IIIA and IIIB of B. cenocepacia strains. We also observed the proximity of samples related to the same patient through the nodes formed in the phylogenetic tree, but the absence of comparison with a reference typing method does not allow to infer their use for epidemiological purposes.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.Submitted by Boris Flegr (boris@uerj.br) on 2021-01-07T15:16:53Z No. of bitstreams: 1 TESE_FINAL_PUBLICADA_Edgard_de_Freitas_Vianna.pdf: 2112429 bytes, checksum: cd95628ce91c98e3e47d66aea8183c15 (MD5)Made available in DSpace on 2021-01-07T15:16:53Z (GMT). No. of bitstreams: 1 TESE_FINAL_PUBLICADA_Edgard_de_Freitas_Vianna.pdf: 2112429 bytes, checksum: cd95628ce91c98e3e47d66aea8183c15 (MD5) Previous issue date: 2017-03-30application/pdfporUniversidade do Estado do Rio de JaneiroPrograma de Pós-Graduação em MicrobiologiaUERJBRCentro Biomédico::Faculdade de Ciências MédicasBurkholderia cepacia complexCystic fibrosisMatrix-Assisted Laser Desorption Ionization-Time of Flight Mass SpectrometryrecA sequencingMatrix Assisted Laser Desorption-IonizationFibrose císticaComplexo Burkholderia cepaciaFibrose císticaSequenciamento do gene recACNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIAIdentificação 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 recAIdentification 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 sequencinginfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UERJinstname:Universidade do Estado do Rio de Janeiro (UERJ)instacron:UERJORIGINALTESE_FINAL_PUBLICADA_Edgard_de_Freitas_Vianna.pdfapplication/pdf2112429http://www.bdtd.uerj.br/bitstream/1/14450/1/TESE_FINAL_PUBLICADA_Edgard_de_Freitas_Vianna.pdfcd95628ce91c98e3e47d66aea8183c15MD511/144502024-02-26 19:54:41.045oai:www.bdtd.uerj.br:1/14450Biblioteca Digital de Teses e Dissertaçõeshttp://www.bdtd.uerj.br/PUBhttps://www.bdtd.uerj.br:8443/oai/requestbdtd.suporte@uerj.bropendoar:29032024-02-26T22:54:41Biblioteca Digital de Teses e Dissertações da UERJ - Universidade do Estado do Rio de Janeiro (UERJ)false |
dc.title.por.fl_str_mv |
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 |
dc.title.alternative.eng.fl_str_mv |
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 |
title |
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 |
spellingShingle |
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 Vianna, Edgard de Freitas Burkholderia cepacia complex Cystic fibrosis Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry recA sequencing Matrix Assisted Laser Desorption-Ionization Fibrose cística Complexo Burkholderia cepacia Fibrose cística Sequenciamento do gene recA CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA |
title_short |
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 |
title_full |
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 |
title_fullStr |
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 |
title_full_unstemmed |
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 |
title_sort |
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 |
author |
Vianna, Edgard de Freitas |
author_facet |
Vianna, Edgard de Freitas |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Marques, Elizabeth de Andrade |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/5959485578597640 |
dc.contributor.referee1.fl_str_mv |
Queiroz, Mara Lucia Penna |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/1980792659825205 |
dc.contributor.referee2.fl_str_mv |
Merquior, Vania Lucia Carreira |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/0742408281665373 |
dc.contributor.referee3.fl_str_mv |
Fírmida, Mônica de Cássia |
dc.contributor.referee3Lattes.fl_str_mv |
http://lattes.cnpq.br/6507048199597970 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/0787358470518689 |
dc.contributor.author.fl_str_mv |
Vianna, Edgard de Freitas |
contributor_str_mv |
Marques, Elizabeth de Andrade Queiroz, Mara Lucia Penna Merquior, Vania Lucia Carreira Fírmida, Mônica de Cássia |
dc.subject.eng.fl_str_mv |
Burkholderia cepacia complex Cystic fibrosis Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry recA sequencing |
topic |
Burkholderia cepacia complex Cystic fibrosis Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry recA sequencing Matrix Assisted Laser Desorption-Ionization Fibrose cística Complexo Burkholderia cepacia Fibrose cística Sequenciamento do gene recA CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA |
dc.subject.por.fl_str_mv |
Matrix Assisted Laser Desorption-Ionization Fibrose cística Complexo Burkholderia cepacia Fibrose cística Sequenciamento do gene recA |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA |
description |
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 by phenotypic methods, molecular methods are recommended. However, there is no universal molecular methodology that can be applied to all microorganisms, especially in clinical laboratories. In this sense, the methodology based on mass spectrometry appears as an alternative. The aim of this study was to evaluate the performance of the Matrix Associated Laser Desorption-Ionization (MALDI-TOF MS) compared to Bcc species identification by recA gene sequencing in CF patients. We analyzed 43 samples obtained from different clinical specimens from January 2014 to February 2016. A total of 18 (41.86%) samples of Burkholderia vietnamiensis, 14 (32.55%) Burkholderia cenocepacia subtype IIIB, eight (18.6%) Burkholderia cenocepacia subtype IIIA, two (4.65%) Burkholderia contaminans and one (2.32%) Burkholderia cepacia were identified by sequencing the recA gene. Identification by MALDI-TOF MS was evaluated considering prior culture in Sheep Blood Agar (SBA) and Columbia Agar Base (CAB). 81.39% of the SBA samples were identified at the genus level (log ≥1.70-1.99) and 41.86% at the species level (log ≥2.00), corresponding to 11 samples of B. vietnamiensis (61.11%), four of B. cenocepacia IIIB (28,57%) and two of B. cenocepacia IIIA (25%). Only 39.53% of the samples cultured in CAB were identified at the genus level and 16.27% in species: B. vietnamiensis (n = 4, 22.22%) and B. cenocepacia IIIB (n = 2; 14,28%). B. cepacia sample was identified from both media and the two B. contaminans samples were not identified. Performance of MALDI-TOF MS varied with the use of different culture media and the identification at the species level was less than 50% considering the cut-off points established by the manufacturer. Thus, we evaluated whether the reduction of cut-off points would increase the accuracy of the identification. With the reduction of the escore to ≥1.70-1.99 (considered as reliable identification at genus level), another three samples of B. vietnamiensis, four of B. cenocepacia IIIA and seven of B. cenocepacia IIIB were identified, corresponding to an increase of 32.55% (in relation to the escore ≥ 2.00). However, the number of samples analyzed does not allow this criterion to be used and additional studies are required. Despite the limitations of MALDI-TOF MS as a typing method, the use of bioinformatics tools to analyze the mass spectra obtained in MALDI-TOF MS allowed the clustering of 96% of the phylogenetic subtypes IIIA and IIIB of B. cenocepacia strains. We also observed the proximity of samples related to the same patient through the nodes formed in the phylogenetic tree, but the absence of comparison with a reference typing method does not allow to infer their use for epidemiological purposes. |
publishDate |
2017 |
dc.date.issued.fl_str_mv |
2017-03-30 |
dc.date.available.fl_str_mv |
2018-01-31 |
dc.date.accessioned.fl_str_mv |
2021-01-07T15:16:53Z |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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dc.identifier.citation.fl_str_mv |
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. 82 f. Dissertação (Mestrado em Microbiologia Médica Humana) - Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2017. |
dc.identifier.uri.fl_str_mv |
http://www.bdtd.uerj.br/handle/1/14450 |
identifier_str_mv |
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. 82 f. Dissertação (Mestrado em Microbiologia Médica Humana) - Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2017. |
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http://www.bdtd.uerj.br/handle/1/14450 |
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Universidade do Estado do Rio de Janeiro |
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Programa de Pós-Graduação em Microbiologia |
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UERJ |
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BR |
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Centro Biomédico::Faculdade de Ciências Médicas |
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Universidade do Estado do Rio de Janeiro |
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