Submitted by Diogo Misoguti (diogo.misoguti@gmail.com) on 2018-04-18T13:18:24Z No. of bitstreams: 1 Tese-15860.pdf: 879437 bytes, checksum: c869f5350ca0a0b0f13be8ba348f8fa5 (MD5) / Approved for entry into archive by Mariusa Loução (mariusa.loucao@unifesp.br) on 2018-04-18T13:20:15Z (GMT) No. of bitstreams: 1 Tese-15860.pdf: 879437 bytes, checksum: c869f5350ca0a0b0f13be8ba348f8fa5 (MD5) / Made available in DSpace on 2018-04-18T13:20:15Z (GMT). No. of bitstreams: 1 Tese-15860.pdf: 879437 bytes, checksum: c869f5350ca0a0b0f13be8ba348f8fa5 (MD5) Previous issue date: 2016 / O uso da biologia molecular como ferramenta de diagnóstico microbiológico vem se expandindo no setor da medicina laboratorial. Novas plataformas aparecem com a prerrogativa de facilitar e acelerar o processo de análise. O uso de sistemas automatizados, que realizam extração, amplificação e detecção de ácidos nucleicos dentro da mesma plataforma, permite maior precisão e facilidade ao desenvolvimento de um novo teste por apresentarem todos os processos acoplados e reagentes disponíveis para o processo. Dentre as plataformas automatizadas, uma das que se destacam é o BD Max™ (Becton Dickinson Diagnostics). O sistema BD Max é uma plataforma automatizada aberta, que combina a extração de ácidos nucleicos, PCR (Reação de Polimerização em Cadeia) em tempo real e detecção dentro do mesmo instrumento, oferecendo a opção de usar os testes aprovados pelo Food and Drug Administration (FDA) e também, testes desenvolvidos pelo usuário. Com o objetivo de testar os recursos que a modalidade aberta da plataforma BD Max oferece, foram desenvolvidos três estudos distintos para o diagnóstico molecular de agentes infecciosos direto de amostras clínicas. Estudo 1: O objetivo deste estudo foi validar um teste multiplex usando a tecnologia PCR em tempo real no sistema aberto BD MAX™, para detectar o complexo Mycobacterium tuberculosis (CMT), complexo Mycobacterium avium (CMA) e Mycobacterium spp. (PAN) diretamente de amostras clínicas. Quando os resultados do novo teste foram comparados com os resultados da cultura, a reação de PCR apresentou especificidade de 97,1%, 100% e 100% para CMT, CMA e PAN, respectivamente. Estudo 2: O objetivo deste estudo foi validar um teste multiplex usando a tecnologia PCR em tempo real no sistema aberto BD MAX™ para detectar o grupo Mycobacterium abscessus (GMA), complexo Mycobacterium fortuitum (CMF) e Mycobacterium chelonae (MC), diretamente de amostras clínicas. Quando os resultados do novo teste foram comparados com os resultados da cultura, uma concordância de 97%, 100% e 99% para GMA, CMF e MC, respectivamente foi observada. Estudo 3: O objetivo deste estudo foi validar um teste multiplex usando a tecnologia PCR em tempo real no sistema aberto BD MAX™ para detectar e identificar Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) e Stenotrophomonas maltophilia (SM) diretamente de amostras respiratórias de pacientes portadores de Fibrose Cística (FC). Quando os resultados do novo teste foram comparados com os resultados da cultura, uma alta concordância foi observada entre as duas metodologias. Conclusão: Os 3 testes desenvolvidos provaram ser específicos e sensíveis para detectar por PCR em tempo real microrganismos causadores de infeção direto da amostra clínica. A plataforma automatizada BD Max provou ser uma excelente ferramenta para a realização de testes moleculares automatizados. / The use of molecular biology as a tool for microbiology diagnostic has been expanding in the laboratory routine. Despite of the strong growth of the area, companies can not afford the demand about epidemiological changes around the world and for this reason laboratories opt to develop their own methods. New platforms appear with the prerogative to facilitate and accelerate the analysis process. The use of automated sample-in results-out platforms allows higher precision and facilitates the development of a new test by presenting all attached processes and reagents available for the test. Among platforms that best fits this profile, the BD Max™ (BD Diagnostics) is one of the most used ones. The BD Max system is an automated open platform that combines extraction and real time PCR in the same instrument, offering the option of using tests approved by the FDA or the open platform mode for userdeveloped test. In order to explore the BD Max open mode platform, three differents studies were developed to detect the microorganisms that causes infection directly from clinical samples. Study 1: A multiplex real time PCR was validated on the BD MAX™ open mode system to detect Mycobacterium tuberculosis complex (MTC), Mycobacterium avium complex (MAC) and Mycobacterium spp. (PAN) directly from clinical specimens. When compared to culture results, the new BD MAX PCR test presented specificities of 97.1%, 100% and 100% for MTC, MAC and PAN, respectively. Study 2: A multiplex real time PCR was validated on the BD MAX™ open mode system to detect Mycobacterium abscesses Group (MAG), Mycobacterium fortuitum complex (MFC) and Mycobacterium chelonae (MC) directly from clinical specimens. When compared to culture results, the new BD Max PCR test presented an overall agreement of 97%, 99% and 100% for the detection of MAG, MFC and MC, respectively. Study 3: A multiplex real time PCR was validated on the BD MAX™ open mode system to detect Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) and Stenotrophomonas maltophilia (SM) directly from clinical specimens collected from Cystic Fibrosis patients. When culture results were compared to the new BD Max PCR test results, a high overall agreement were observed between both methodologies. Conclusion: All 3 tests proved to be specific and sensitive to detect different microorganisms associated with infections, directly from clinical samples. The BD Max proved to be an excellent tool for automated molecular tests.

Background: Vancomycin-resistant enterococci (VRE) is a common nosocomial infection. It classifies as Enterococcus faecalis or faecium carrying vanA or vanB gene that alters the bacterial cell wall hence lowering affinity for Vancomycin. Screening for VRE in Swedish hospitals are performed with stool sample pre-grown in selective broth followed by PCR and culture on selective media. Viasures Vancomycin resistance, Real Time PCR Detection Kit indicates that pre-growth in broth is not needed for the analyze. Aim: Comparison between the PCR kit and the subsequent culture on chromogenic agar with or without pre-growth in selective broth. Method: E. faecium with vanA gen (CCUG 36804) and E. faecalis with vanB gen (ATCC 51299) were suspended in different concentrations and added to ESwab transport medium. Thereafter small samples from the ESwab tube were enriched in selective broth. Samples from both selective broth and ESwab medium were analyzed with Viasures PCR kit on BD-MAX system and cultivated on chromogenic agar. Results: With pre-growth in selective broth the genes were found in every sample regardless of pre-concentration in the ESwab medium. Without enrichment the PCR kit always amplified the genes when the concentration was 40 000 cfu/ml for E. faecium (vanA) and ≥ 10 000 cfu/ml for E. faecalis (vanB). Colonies grew on chromogenic agar in every concentration from both ESwab and selective broth. Conclusion: Culture on chromogenic agar is comparable with or without pre-growth in selective broth but Viasure’s PCR kit is not equal for both methods in lower concentrations of the bacteria.