Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides
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 identi...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Fundação Oswaldo Cruz (FIOCRUZ)
2020-04-01
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| Series: | Memorias do Instituto Oswaldo Cruz |
| Subjects: | |
| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762020000100309&tlng=en |
| _version_ | 1797721313820803072 |
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| author | Graziele Lima Bello Franciele Costa Leite Morais Sheile Pinheiro de Jesus Jonas Michel Wolf Mirela Gehlen Isabela Neves de Almeida Lida Jouca de Assis Figueiredo Tainá dos Santos Soares Regina Bones Barcellos Elis Regina Dalla Costa Silvana Spíndola de Miranda Maria Lucia Rosa Rossetti |
| author_facet | Graziele Lima Bello Franciele Costa Leite Morais Sheile Pinheiro de Jesus Jonas Michel Wolf Mirela Gehlen Isabela Neves de Almeida Lida Jouca de Assis Figueiredo Tainá dos Santos Soares Regina Bones Barcellos Elis Regina Dalla Costa Silvana Spíndola de Miranda Maria Lucia Rosa Rossetti |
| author_sort | Graziele Lima Bello |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T09:31:41Z |
| format | Article |
| id | doaj.art-f0ef69fb32364e199a9b4657121c8135 |
| institution | Directory Open Access Journal |
| issn | 1678-8060 |
| language | English |
| last_indexed | 2024-03-12T09:31:41Z |
| publishDate | 2020-04-01 |
| publisher | Fundação Oswaldo Cruz (FIOCRUZ) |
| record_format | Article |
| series | Memorias do Instituto Oswaldo Cruz |
| spelling | doaj.art-f0ef69fb32364e199a9b4657121c81352023-09-02T13:54:12ZengFundação Oswaldo Cruz (FIOCRUZ)Memorias do Instituto Oswaldo Cruz1678-80602020-04-0111510.1590/0074-02760190407Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slidesGraziele Lima Bellohttps://orcid.org/0000-0001-8537-8413Franciele Costa Leite MoraisSheile Pinheiro de JesusJonas Michel WolfMirela GehlenIsabela Neves de AlmeidaLida Jouca de Assis FigueiredoTainá dos Santos SoaresRegina Bones BarcellosElis Regina Dalla CostaSilvana Spíndola de MirandaMaria Lucia Rosa RossettiBACKGROUND 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.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762020000100309&tlng=enMycobacterium tuberculosisIsoniazid resistancegenotypingreal-time PCR |
| spellingShingle | Graziele Lima Bello Franciele Costa Leite Morais Sheile Pinheiro de Jesus Jonas Michel Wolf Mirela Gehlen Isabela Neves de Almeida Lida Jouca de Assis Figueiredo Tainá dos Santos Soares Regina Bones Barcellos Elis Regina Dalla Costa Silvana Spíndola de Miranda Maria Lucia Rosa Rossetti Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides Memorias do Instituto Oswaldo Cruz Mycobacterium tuberculosis Isoniazid resistance genotyping real-time PCR |
| title | Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides |
| title_full | Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides |
| title_fullStr | Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides |
| title_full_unstemmed | Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides |
| title_short | Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides |
| title_sort | rapid detection of mycobacterium tuberculosis dna and genetic markers for isoniazid resistance in ziehl neelsen stained slides |
| topic | Mycobacterium tuberculosis Isoniazid resistance genotyping real-time PCR |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762020000100309&tlng=en |
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