title: Real time PCR quantification of viable Mycobacterium tuberculosis from sputum samples treated with propidium monoazide.
abstract: Diagnostic methods of TB, nowadays, are prone to delay in diagnosis, increased false negative results and are not sensitive to many forms of paucibacillary disease. The aims of this study were to implement a quantitative nucleic acid-based diagnostic test for paucibacillary tuberculosis, enabling the identification and quantification of viable Mycobacterium tuberculosis bacilli by quantitative Real-Time PCR (qRT-PCR). The intergenic region of the single-copy inhA-mabA gene was chosen as the target region for design of primers and probes conjugated with fluorophores. The construction of synthetic DNA flanking the target region served as standards for absolute quantification of nucleic acids. Using the intercaling dye, propidium monoazide, we were able to discriminate between viable and dead cells of M. tuberculosis. The diagnosis method showed a broad sensitivity (96.1%) when only compared to samples of smear-positive sputum and ROC analyses shows that our approach performed well and yielded a specificity of 84.6% and a sensitivity of 84.6% when compared to M. tuberculosis colony-forming units counting.
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul - PUCRS, Avenida Ipiranga, 6681, TecnoPUC 92A, 90619-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS, Porto Alegre, RS, Brazil.
authors.forename: Thiago Milech
authors.initials: TM
authors.lastname: de Assunção
authors.affiliation: Faculdade de Odontologia, PUCRS, Porto Alegre, RS, Brazil.
authors.forename: Eraldo L
authors.initials: EL
authors.lastname: Batista
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul - PUCRS, Avenida Ipiranga, 6681, TecnoPUC 92A, 90619-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Medicina e Ciências da Saúde, PUCRS, Porto Alegre, RS, Brazil.
authors.forename: Candida
authors.initials: C
authors.lastname: Deves
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul - PUCRS, Avenida Ipiranga, 6681, TecnoPUC 92A, 90619-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Medicina e Ciências da Saúde, PUCRS, Porto Alegre, RS, Brazil.
authors.forename: Anne Drumond
authors.initials: AD
authors.lastname: Villela
authors.affiliation: Faculdade de Farmácia, PUCRS, Porto Alegre, RS, Brazil; Hospital São Lucas, PUCRS, Porto Alegre, RS, Brazil.
authors.forename: Vany Elisa
authors.initials: VE
authors.lastname: Pagnussatti
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul - PUCRS, Avenida Ipiranga, 6681, TecnoPUC 92A, 90619-900 Porto Alegre, RS, Brazil.
authors.forename: Ana Christina
authors.initials: AC
authors.lastname: de Oliveira Dias
authors.affiliation: Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Afrânio
authors.initials: A
authors.lastname: Kritski
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul - PUCRS, Avenida Ipiranga, 6681, TecnoPUC 92A, 90619-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Medicina e Ciências da Saúde, PUCRS, Porto Alegre, RS, Brazil. Electronic address: valnes.rodrigues@acad.pucrs.br.
authors.forename: Valnês
authors.initials: V
authors.lastname: Rodrigues-Junior
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul - PUCRS, Avenida Ipiranga, 6681, TecnoPUC 92A, 90619-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS, Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Medicina e Ciências da Saúde, PUCRS, Porto Alegre, RS, Brazil.
authors.forename: Luiz Augusto
authors.initials: LA
authors.lastname: Basso
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul - PUCRS, Avenida Ipiranga, 6681, TecnoPUC 92A, 90619-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS, Porto Alegre, RS, Brazil. Electronic address: diogenes@pucrs.br.
authors.forename: Diógenes Santiago
authors.initials: DS
authors.lastname: Santos
mesh_terms: D000345:Affinity Labels; D001386:Azides / Q000008:administration & dosage / Q000494:pharmacology*; D015169:Colony Count, Microbial; D004396:Coloring Agents / Q000008:administration & dosage / Q000494:pharmacology; D004269:DNA, Bacterial / Q000032:analysis; D021901:DNA, Intergenic / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D006801:Humans; D050296:Microbial Viability; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D011419:Propidium / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis*
chemical_list: D000345:Affinity Labels; D001386:Azides; D004396:Coloring Agents; D004269:DNA, Bacterial; D021901:DNA, Intergenic; C533957:propidium monoazide; D011419:Propidium; C014535:8-azidoethidium
keywords: Diagnosis; PMA; Real time PCR; Smear; Tuberculosis