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

Submitted by Kamylla Nascimento ([email protected]) on 2017-12-12T12:54:02Z No. of bitstreams: 1 art. Rapid detection and - lima.pdf: 783790 bytes, checksum: 3b012d008462813b5ebe455b2f2f2e83 (MD5)Approved for entry into archive by Kamylla Nascimento ([email protected]) on 2017-12-12T13:06:12Z (GMT) No. of bitstreams: 1 art. Rapid detection and - lima.pdf: 783790 bytes, checksum: 3b012d008462813b5ebe455b2f2f2e83 (MD5)Made available in DSpace on 2017-12-12T13:06:13Z (GMT). No. of bitstreams: 1 art. Rapid detection and - lima.pdf: 783790 bytes, checksum: 3b012d008462813b5ebe455b2f2f2e83 (MD5) Previous issue date: 2013Instituto Aggeu Magalhães (Fiocruz-PE).Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de imunologia. Recife, PE, Brasil.Universidade Federal do Rio Janeiro. Instituto de Microbiologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de imunologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de imunologia. Recife, PE, Brasil.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

Desempenho da técnica nested PCR na detecção específica do complexo Mycobacterium tuberculosis em amostras sanguíneas de pacientes pediátricos

OBJETIVO: Avaliar o desempenho da técnica nested PCR (nPCR) para detectar o complexo Mycobacterium tuberculosis em amostras de sangue de pacientes com suspeita de TB para sua possível utilização como uma ferramenta auxiliar no diagnóstico laboratorial da doença em crianças. MÉTODOS: Detecção do complexo M. tuberculosis em amostras de sangue usando como alvo a sequência de inserção IS6110 do DNA genômico do bacilo. Foram avaliados 120 pacientes, menores de 15 anos de idade, de ambos os sexos, provenientes de hospitais públicos do Recife (PE), no período entre janeiro de 2003 e agosto de 2005. O diagnóstico de TB foi realizado pelo médico assistente do serviço de saúde de acordo com os critérios da Sociedade Torácica Americana. A nPCR amplificou um fragmento de 123 pb com oligonucleotídeos externos (IS1/IS2) e, na reação subsequente, com oligonucleotídeos internos (IS3/IS4), gerando um amplicon de 81 pb. RESULTADOS: A TB ativa ou latente esteve presente em 65 pacientes, foi descartada em 28 suspeitos e 27 não tinham a doença (controles). A sensibilidade da nPCR foi de 26,15%, sendo significativamente maior na forma extrapulmonar (55,56%) em relação à pulmonar (18,18%), e a especificidade foi de 92,73%. CONCLUSÕES: Diante das dificuldades diagnósticas da TB infantil e do baixo número de casos estudados, a nPCR em sangue demonstrou ser uma técnica rápida e específica, mas com baixa sensibilidade. Para saber a sua real utilidade no diagnóstico de formas paucibacilares, sobretudo as extrapulmonares, novas pesquisas devem ser desenvolvidas com uma casuística maior de crianças e com outros espécimes biológicos além do sangue

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples

Submitted by Ana Beatriz Oliveira ([email protected]) on 2019-04-22T13:04:15Z No. of bitstreams: 1 Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples..pdf: 1052057 bytes, checksum: 7aee4b7c7f3946a2f4d18ba85f388dfb (MD5)Approved for entry into archive by Ana Beatriz Oliveira ([email protected]) on 2019-04-22T13:30:48Z (GMT) No. of bitstreams: 1 Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples..pdf: 1052057 bytes, checksum: 7aee4b7c7f3946a2f4d18ba85f388dfb (MD5)Made available in DSpace on 2019-04-22T13:30:48Z (GMT). No. of bitstreams: 1 Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples..pdf: 1052057 bytes, checksum: 7aee4b7c7f3946a2f4d18ba85f388dfb (MD5) Previous issue date: 2018Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Imunologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Imunologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Imunologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Imunologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Imunologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Imunologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Imunologia. Recife, PE, Brasil.Introduction: This study evaluated the performance of the IS6110-TaqMan® assay in different types of biological samples and tissues for laboratory diagnosis of extrapulmonary tuberculosis. Methods: 143 biological samples and tissues from patients with suspected extrapulmonary tuberculosis from the health services of Recife/Pernambuco/Brazil were evaluated with the IS6110-TaqMan® assay. Results: The sensitivities of the IS6110-TaqMan® assay calculated for blood, urine, both blood and urine samples, tissue biopsies, extrapulmonary body fluid samples, and all samples from patients calculated together were 55.9%, 33.3%, 68.8%, 43.8%, 29.6%, and 73.7%, respectively, and the specificities were 80%, 100%, 78.6%, 100%, 100%, and 84.2%, respectively. Conclusions: The accuracy of qPCR was high in various clinical sample types. The analysis of more than one type of clinical sample collected from the same patient with extrapulmonary tuberculosis enhances the diagnostic power of the IS6110-TaqMan® assay when compared with the use of only one clinical sample

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples

Abstract INTRODUCTION: This study evaluated the performance of the IS6110-TaqMan® assay in different types of biological samples and tissues for laboratory diagnosis of extrapulmonary tuberculosis. METHODS: 143 biological samples and tissues from patients with suspected extrapulmonary tuberculosis from the health services of Recife/Pernambuco/Brazil were evaluated with the IS6110-TaqMan® assay. RESULTS: The sensitivities of the IS6110-TaqMan® assay calculated for blood, urine, both blood and urine samples, tissue biopsies, extrapulmonary body fluid samples, and all samples from patients calculated together were 55.9%, 33.3%, 68.8%, 43.8%, 29.6%, and 73.7%, respectively, and the specificities were 80%, 100%, 78.6%, 100%, 100%, and 84.2%, respectively. CONCLUSIONS The accuracy of qPCR was high in various clinical sample types. The analysis of more than one type of clinical sample collected from the same patient with extrapulmonary tuberculosis enhances the diagnostic power of the IS6110-TaqMan® assay when compared with the use of only one clinical sample

Diferenciação de micobactérias por PCR multiplex

O trabalho visou à otimização de um método baseado na reação em cadeia da polimerase multiplex - para diferenciação de micobactérias de interesse para a saúde pública. A PCR Multiplex baseou-se na amplificação simultânea do genehsp65, presente em todo gênero Mycobacterium, do gene dnaJ, presente apenas em Mycobacterium tuberculosis e Mycobacterium avium e da sequência de inserção IS6110 presente no complexo Mycobacterium tuberculosis, gerando amplicons de 165pb, 365pb e 541pb, respectivamente. O limite de detecção foi de 1fg para o alvo hsp65, 100pg para o dnaJ e 0,1fg para o IS6110. A PCR multiplex detectou até 100pg de DNA de Mycobacterium tuberculosis. O sistema demonstrou ser específico e sensível na detecção de Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium e Mycobacterium smegmatis. Os resultados obtidos utilizando cepas de referência demonstraram que a PCR multiplex pode ser uma ferramenta rápida, sensível e específica na diferenciação de micobactérias

Evaluation of a nested-pcr for Mycobacterium tuberculosis detection in blood and urine samples

The polymerase chain reaction (PCR) and its variations, such as the nested-PCR, have been described as promising techniques for rapid diagnosis of tuberculosis (TB). With the aim of evaluating the usefulness of a nested-PCR method on samples of blood and urine of patients suspected of tuberculosis we analyzed 192 clinical samples, using as a molecular target the insertion element IS6110 specific of M. tuberculosis genome. Nested-PCR method showed higher sensitivity in patients with extrapulmonary tuberculosis (47.8% and 52% in blood and urine) when compared to patients with the pulmonary form of the disease (sensitivity of 29% and 26.9% in blood and urine), regardless of the type of biological sample used. The nested-PCR is a rapid technique that, even if not showing a good sensitivity, should be considered as a helpful tool especially in the extrapulmonary cases or in cases where confirmatory diagnosis is quite difficult to be achieved by routine methods. The performance of PCR-based techniques should be considered and tested in future works on other types of biological specimens besides sputum, like blood and urine, readily obtainable in most cases. The improving of M. tuberculosis nested-PCR detection in TB affected patients will give the possibility of an earlier detection of bacilli thus interrupting the transmission chain of the disease

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses

Submitted by Kamylla Nascimento ([email protected]) on 2017-12-11T12:03:54Z No. of bitstreams: 1 art. The Performance - montenegro.pdf: 788710 bytes, checksum: 78b6665e69363b833df61e5eb1eb9977 (MD5)Approved for entry into archive by Kamylla Nascimento ([email protected]) on 2017-12-11T12:20:35Z (GMT) No. of bitstreams: 1 art. The Performance - montenegro.pdf: 788710 bytes, checksum: 78b6665e69363b833df61e5eb1eb9977 (MD5)Made available in DSpace on 2017-12-11T12:20:35Z (GMT). No. of bitstreams: 1 art. The Performance - montenegro.pdf: 788710 bytes, checksum: 78b6665e69363b833df61e5eb1eb9977 (MD5) Previous issue date: 2013Este estudo recebeu apoio financeiro do Centro de Pesquisa Aggeu Magalhães - FIOCRUZ e do Programa de Desenvolvimento Tecnológico em Suprimentos de Saúde (PDTIS).Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de imunologia. Recife, PE, Brasil.Hospital Barão de Lucena. Departamento de Clínica Médica. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de imunologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de imunologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de imunologia. Recife, PE, Brasil.Hospital Geral Otávio de Freitas. Recife, PE, Brasil.Hospital Barão de Lucena. Departamento de Clínica Médica. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de imunologia. Recife, PE, Brasil.This study evaluated the performance of an in-house nested-PCR system for the detection of the Mycobacterium tuberculosis complex in pleural fluid, blood and urine samples from pleural effusion tuberculosis patients by health services physicians in Pernambuco, Brazil