title: In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA.
abstract: Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.
authors.affiliation: Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde, Porto Alegre, RS, Brasil.
authors.forename: Sergio Luiz Montego
authors.initials: SL
authors.lastname: Ferreira Junior
authors.affiliation: Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde, Porto Alegre, RS, Brasil.
authors.forename: Elis Regina
authors.initials: ER
authors.lastname: Dalla Costa
authors.affiliation: Instituto de Doenças do Tórax, Hospital Universitário Clementino Fraga Filho, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
authors.forename: Paula Gonçalves dos
authors.initials: PG
authors.lastname: Santos
authors.affiliation: Laboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil.
authors.forename: Harrison Magdinier
authors.initials: HM
authors.lastname: Gomes
authors.affiliation: Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde, Porto Alegre, RS, Brasil.
authors.forename: Marcia Susana Nunes
authors.initials: MS
authors.lastname: Silva
authors.affiliation: Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde, Porto Alegre, RS, Brasil.
authors.forename: Leonardo Souza
authors.initials: LS
authors.lastname: Esteves
authors.affiliation: Instituto de Doenças do Tórax, Hospital Universitário Clementino Fraga Filho, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
authors.forename: Martha Maria
authors.initials: MM
authors.lastname: Oliveira
authors.affiliation: Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde, Porto Alegre, RS, Brasil.
authors.forename: Raquel de Abreu
authors.initials: Rde A
authors.lastname: Maschmann
authors.affiliation: Instituto de Doenças do Tórax, Hospital Universitário Clementino Fraga Filho, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
authors.forename: Afrânio Lineu
authors.initials: AL
authors.lastname: Kritski
authors.affiliation: Laboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil.
authors.forename: Philip Noel
authors.initials: PN
authors.lastname: Suffys
authors.affiliation: Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde, Porto Alegre, RS, Brasil.
authors.forename: Maria Lucia Rosa
authors.initials: ML
authors.lastname: Rossetti
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D002374:Catalase / Q000235:genetics*; D003124:Colorimetry; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D060005:Genotyping Techniques; D007538:Isoniazid / Q000494:pharmacology; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010088:Oxidoreductases / Q000235:genetics*; D012293:Rifampin / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; C085257:InhA protein, Mycobacterium; D012321:DNA-Directed RNA Polymerases; D007538:Isoniazid; D012293:Rifampin