Mem Inst Oswaldo Cruz, Rio de Janeiro, VOLUME 115 | MARCH 2020
Original Article

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

Graziele Lima Bello1/+, Franciele Costa Leite Morais1, Sheile Pinheiro de Jesus1, Jonas Michel Wolf1, Mirela Gehlen2, Isabela Neves de Almeida3, Lida Jouca de Assis Figueiredo3, Tainá dos Santos Soares4, Regina Bones Barcellos5,6, Elis Regina Dalla Costa6,7, Silvana Spíndola de Miranda3, Maria Lucia Rosa Rossetti1,4,5,6

1Universidade Luterana do Brasil, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Canoas, RS, Brasil
2Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Pneumologia, Porto Alegre, RS, Brasil
3Universidade Federal de Minas Gerais, Faculdade de Medicina, Laboratório de Pesquisa em Micobactérias, Belo Horizonte, MG, Brasil
4Universidade Luterana do Brasil, Graduação em Biomedicina, Canoas, RS, Brasil
5Secretaria do Estado do Rio Grande do Sul, Centro de Desenvolvimento Científico e Tecnológico, Porto Alegre, RS, Brasil
6Universidade Federal do Rio de Janeiro, Programa de Pós-Graduação em Clínica Médica, Rio de Janeiro, RJ, Brasil
7AstraZeneca do Brasil, Cotia, SP, Brasil

DOI: 10.1590/0074-02760190407
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ABSTRACT

BACKGROUND 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.

OBJECTIVES 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.

METHODS 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.

FINDINGS 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.

MAIN CONCLUSIONS 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.

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GB and JMW were supported by CAPES (MEC/Brazil-Fellowship. Financing code 001).
+ Corresponding author: grazilbello@gmail.com
 https://orcid.org/0000-0001-8537-8413
Received 31 October 2019
Accepted 06 March 2020

CITATION: Bello GL, Morais FCL, de Jesus SP, Wolf JM, Gehlen M, de Almeida IN, et al. Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides. Mem Inst Oswaldo Cruz. 2020; 115: e190407.

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