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

• Autor(a) principal: Bello,Graziele Lima
• Data de Publicação: 2020
• Outros Autores: 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
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Memórias do Instituto Oswaldo Cruz
• Texto Completo: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762020000100309
• topic: Mycobacterium tuberculosis; Isoniazid resistance; genotyping; real-time PCR

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.