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.
Highlights
Diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to antiTB drugs are considered the main factors for disease control
Standardisation of TaqMan® qPCR genotyping assays to the detection of INH resistance in culture of M. tuberculosis - This study was performed using a total of 55 independent DNA samples extracted from clinical isolates of M. tuberculosis, according to described by Van Soolingen et al,(10) Samples were stored at -80oC in the Molecular Biology Laboratory, at the Centre for Scientific and Technological Development (CDCT) of the State Health Secretariat (SES), located in the city of Porto Alegre, Southern Brazil
All the 55 isolates used in the present study were confirmed as containing acid-fast bacilli by microscopy detection on ZN-stained slides.[11]. Standard bacteriological and biochemical tests were performed for differentiation of species within the M. tuberculosis complex (MTBC) and Mycobacteria other than tuberculosis (MOTT), including biochemical testing for niacin, paranitrobenoic acid (PNB) and tiofeno-2-carboxylic acid hydrazine (TCH).(11) Subsequently, the isolates were submitted to drug susceptibility testing (DST) using the BactecTMMGITTM960 system, according to the manufacturer.[12]
Summary
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
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