Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides

Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in...

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Published in:	Memórias do Instituto Oswaldo Cruz Vol. 115; p. e190407
Main Authors:	Bello, Graziele Lima, Morais, Franciele Costa Leite, de Jesus, Sheile Pinheiro, Wolf, Jonas Michel, Gehlen, Mirela, de Almeida, Isabela Neves, Figueiredo, Lida Jouca de Assis, Soares, Tainá Dos Santos, Barcellos, Regina Bones, Dalla Costa, Elis Regina, de Miranda, Silvana Spíndola, Rossetti, Maria Lucia Rosa
Format:	Journal Article
Language:	English
Published:	Brazil Instituto Oswaldo Cruz, Ministério da Saúde 01-01-2020 Fundação Oswaldo Cruz (FIOCRUZ)
Subjects:	Antitubercular Agents - pharmacology DNA, Bacterial - genetics Drug Resistance, Bacterial - genetics Genetic Markers - genetics Genotype genotyping Humans Isoniazid - pharmacology Isoniazid resistance Microbial Sensitivity Tests Mutation - genetics Mycobacterium tuberculosis Mycobacterium tuberculosis - drug effects Mycobacterium tuberculosis - genetics Original PARASITOLOGY real-time PCR Real-Time Polymerase Chain Reaction Sensitivity and Specificity TROPICAL MEDICINE Isoniazid resistance real-time PCR Mycobacterium tuberculosis genotyping
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Summary:	Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 GLB - Lead author, participating from the experimental phase, results from analysis and scientific writing of the manuscript; FCLM - contributed with the experimental phase of the standardisation steps until the application of the technique in smear slides; MG - contributed to the initial stage of the experiments; SPJ - participated in the experimental stage of application of the technique; JMW - contributed statistical analysis and scientific writing of the manuscript; INA - contributed to the analyses performed at the Federal University of Minas Gerais (Brazil) and the scientific writing of the manuscript; LJAF - contributed to the analyses carried out at the Federal University of Minas Gerais (Brazil) and the scientific writing of the manuscript; TSS - contributed to the final stage of the experimental analyses; ERDC - contributed to the scientific writing of the manuscript; RBB - contributed with the experimental phase, results from analysis and scientific writing of the manuscript, as well as the supply of the Mycobacterium tuberculosis strains sample bank for the technique standardisation stage; SSM - co-advisor, contributed to the analyses performed at the Federal University of Minas Gerais (Brazil) and the scientific writing of the manuscript, as well as the supply of slides for analysis; MLRR - study advisor, responsible for the elaboration and application of the project, as well as the scientific writing of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
ISSN:	0074-0276 1678-8060 1678-8060
DOI:	10.1590/0074-02760190407