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BACKGROUND Non-tuberculous mycobacteria (NTMs) cause diseases known as mycobacteriosis and are an important cause of morbidity and mortality. The diagnosis of pulmonary disease caused by NTM is hampered by its clinical similarity with tuberculosis (TB) and by the lack of an accurate and rapid laboratory diagnosis. OBJECTIVES Detect DNA from NTMs directly from lung samples using real-time polymerase chain reaction (qPCR) for amplification of 16S rRNA. Additionally, DNA sequencing ( hsp65 and rpoB genes) was used to identify the species of MNTs. METHODS A total of 68 sputum samples (54 with suspected NTMs and 14 with TB) from patients treated at a referral hospital were used. FINDINGS Of these, 27/54 (50%) were qPCR positive for NTMs and 14/14 TB patients (controls) were qPCR negative with an almost perfect concordance ( Kappa of 0.93) with the Mycobacterium spp. culture. Sequencing confirmed the presence of NTM in all positive samples. The most common species was Mycobacterium gordonae (33%), followed by Mycobacterium abscessus (26%), Mycobacterium fortuitum (22%), Mycobacterium avium (15%) and Mycobacterium peregrinum (4%). MAIN CONCLUSIONS The qPCR technique for detecting NTMs targeting 16S rRNA has the potential to detect NTMs and rapidly differentiate from Mycobacterium tuberculosis . However, it is necessary to identify the species to help in the differential diagnosis between disease and contamination, and to guide the choice of the therapeutic scheme.

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 ZNstained 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.