Diferenciação de micobactérias por PCR multiplex

Poroca, Diogo da Rocha; Lima, Andrea Santos; Lima, Juliana Falcão de Araújo; Cruz, Heidi Lacerda Alves da; Montenegro, Rosana de Albuquerque; Melo, Fábio Lopes de; Schindler, Haiana Charifker; Montenegro, Lílian Maria Lapa

doi:10.1590/s0037-86822009000600020

Cited by 9 publications

(20 citation statements)

References 28 publications

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“…None of the results indicated the presence of the M. avium complex or a fi nal volume of 50µL. These reaction parameters are in accordance with the conditions described by Poroca et al 26 .…”

Section: Species Identifi Cation Using Restriction Enzyme Analysissupporting

confidence: 89%

“…In 13 of the samples (32.5%), amplifi cation of a fragment sized 20 to 40 base pairs less than 165bp was observed, which characterized these samples as NTM, according to Poroca et al 26 ( Figure 1). M. intracellulare among the strains isolated via culture from the pulmonary and extrapulmonary tuberculosis samples.…”

Section: Evaluation Of Multiplex Pcr Using Mycobacterial Speciesmentioning

confidence: 79%

“…Multiplex PCR has the ability to amplify different targets simultaneously and has been used to detect and identify mycobacteria from the Mycobacterium tuberculosis complex and nontuberculous mycobacteria 26,27,35,36 . In this study, when the multiplex PCR was compared with the reference tests (cultures and phenotypic and biochemical tests) to identify 40 mycobacterial isolates, a signifi cant agreement was observed: 22 (100%) of the 37 cases with a fi nal diagnoses of pulmonary or extrapulmonary tuberculosis presented species of the Mycobacterium tuberculosis complex (MTBC) using the reference tests.…”

Section: Discussionmentioning

confidence: 99%

“…This system, which amplifies two or three different targets, can differentiate M. tuberculosis from the NTMs 25,26 . This molecular approach is used in identifying and differentiating microbes because it is able to supply a simple fi ngerprint of certain bacterial groups when compared to the standard profi les of referent strains.…”

Section: Methodsmentioning

confidence: 99%

“…The correct choice of target sequences in the genome is one of the key criteria for detecting and identifying mycobacteria by PCR 27 . Faced with the need to differentiate tuberculosis from other mycobacteria, the aim of this study was to evaluate a system based on multiplex PCR, already optimized by Poroca et al 26 , for identifying mycobacterial species isolated from different clinical samples from patients diagnosed with pulmonary and extrapulmonary tuberculosis. This test, in addition to contributing to a rapid and accurate differential diagnosis between tuberculosis and other mycobacteria, will certainly assist in the early and appropriate therapeutic management of the patient.…”

Section: Methodsmentioning

confidence: 99%

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Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Lima

Duarte

Montenegro

et al. 2013

Rev. Soc. Bras. Med. Trop.

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Introduction:The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods: The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) Results: Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identifi cation of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the fi nal diagnosis from the attending physician. Conclusions: Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifi able using conventional biochemical and phenotypic techniques.

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Section: Species Identifi Cation Using Restriction Enzyme Analysissupporting

confidence: 89%

Section: Evaluation Of Multiplex Pcr Using Mycobacterial Speciesmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Lima

Duarte

Montenegro

et al. 2013

Rev. Soc. Bras. Med. Trop.

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Desenvolvimento de micobacteriófago recombinante para detecção rápida de bacilos da tuberculose

Silva¹

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Aos meus pais, Manoel Lucas da Silva e Maria José da Silva, que lutaram bravamente pela educação de todos os filhos, prestando apoio e amor incondicional em todos os momentos da minha vida. A minha esposa, Fábia de Oliveira Andrade, pelo amor, compreensão e união, que foram fundamentais na realização deste sonho. Aos meus irmãos, pelo incentivo na busca de meus sonhos. AGRADECIMENTOS A Deus, por estar sempre comigo e guiar o meu caminho. Ao meu orientador, Prof. Tit. Mario Hiroyuki Hirata, pela orientação deste trabalho e ao singular estímulo ao livre pensamento científico. À Prof. Tit. Rosário Dominguez Crespo Hirata, pela paciência, críticas e sugestões no desenvolvimento deste e de outros projetos. Ao prof. Graham F. Hatfull pela grande oportunidade em desenvolver parte fundamental dos experimentos em seu laboratório sob a sua orientação e pelas valiosas informações sobre clonagem e recombinação em micobactérias e fagos. À Prof. Mariana Piuri, pelo estimado apoio e valiosos ensinamentos prestados durante a minha passagem pela Universidade de Pittsburgh. À Cristina Moreno Fajardo, técnica do Laboratório de Biologia Molecular Aplicada ao Diagnóstico da FCF/USP, pela amizade, ensinamentos, e apoio técnico fundamentais no desenvolvimento deste trabalho. Aos Prof. Elsa Masae Mamizuka e Jorge Luiz Mello Sampaio pelas sugestões nas inúmeras tentativas efetuadas para superar os obstáculos que surgiram ao longo deste projeto. Aos meus pais, Manoel Lucas da Silva (in memoriam) e Maria José da Silva, e aos meus irmãos Maria do Carmo Silva Pereira, Carmen Lúcia Silva, Rinaldo Lucas da Silva e João Roberto da Silva por todo apoio e compreensão prestados durante todo este trabalho. A minha maravilhosa esposa, Fábia de Oliveira Andrade, por sempre estar ao meu lado e incentivar a busca pela realização de nossos sonhos. Aos colegas do laboratório, especialmente Gisele de Medeiros Bastos, Walkíria de Araújo Souza, e Gabriela Guimarães Sousa Leite, pelo apoio científico e grande amizade. A todos que compõem a Faculdade de Ciências Farmacêuticas da Universidade de São Paulo e o Departamento de Análises Clínicas e toxicológicas pela oportunidade de realização do curso de Doutorado. A Fundação de Amparo a Pequisa do Estado de São Paulo (FAPESP), pelos recursos financeiros disponibilizados para este projeto sob o n. 2008/05076-7 e pela bolsa de doutorado concedida sob o n. 2008/05082-7. RESUMO SILVA, J.L. Desenvolvimento de micobacteriófago recombinante para detecção rápida de bacilos da tuberculose. Tese (Doutorado)

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Diagnosis of mycobacterial infections based on acid-fast bacilli test and bacterial growth time and implications on treatment and disease outcome

et al. 2016

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BackgroundThe establishment of therapeutic regimens for mycobacteriosis depends on the accurate identification of Mycobacterium species, and misdiagnosis can result in inappropriate treatment and increased mortality of patients. Differential diagnosis among Mycobacterium species has been made by conventional phenotypic and biochemical tests after a long culture period. Specialized molecular diagnostics of mycobacteria allows rapid detection and species identification; however, such tests are not available in public health programs. Our aim was to demonstrate the clinical implications of erroneous diagnosis by performing molecular genotyping of mycobacterial infections in patients that were diagnosed based on symptoms, culture and bacilloscopy.MethodsCulture samples of mycobacterial infections from 55 patients clinically diagnosed as tuberculosis in 2013 and 2014, based on conventional methods, were identified by PCR -RFLP and results are discussed.ResultsWe have confirmed 35 (63.6 %) positive samples as M. tuberculosis, but 18 (32.7 %) were identified as non-tuberculous mycobacteria (M. avium type 1, M. avium type 2, M. kansasii type 1 type 1, M. mucogenicum, M. chelonae, M. terrae type 3, and 1 unknown RFLP pattern) and two were negative. Regarding clinical diagnosis, 61.8 % (34/55) was classified as pulmonary tuberculosis. It is important to emphasize that 36.4 % (20/55) of samples were misdiagnosed by conventional methods, and 11 (61.1 %) of the HIV positive patients (18/55) were NTM-coinfected.ConclusionThe identification of species in mycobacterial infections is essential for correct diagnosis and choice of treatment regimen, and misdiagnosis by conventional tools can lead to chronic disease, increased resistance and death.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1474-6) contains supplementary material, which is available to authorized users.

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Diferenciação de micobactérias por PCR multiplex

Cited by 9 publications

References 28 publications

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Desenvolvimento de micobacteriófago recombinante para detecção rápida de bacilos da tuberculose

Diagnosis of mycobacterial infections based on acid-fast bacilli test and bacterial growth time and implications on treatment and disease outcome

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