Detection of non-tuberculosus mycobacteria (NTMs) in lung samples using 16S rRNA

Abstract

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.

Clinical and Pathological Findings Associated with Mycobacteriosis in Captive Syngnathids

“…In fact, the 16s rRNA, hsp65 and rpoB gene sequences have a similarity of 97% [ 28 ]. However, other studies [ 29 ] found that the 16s rRNA is useful for identifying NTM species and differentiating them from the rpoB gene of Mycobacterium complex by PCR. Previous literature describes those seahorses, seadragons and pipefish commonly suffer significant losses primarily due to M chelonae [ 3 , 11 , 12 ]; however, that was not found in this study, leading to the conclusion that it seems not be an important pathogen in the aquatic facilities where the studied animals were maintained.…”

Rapid and accurate identification and differentiation of Mycobacterium tuberculosis and non-tuberculous mycobacteria using PCR kits available in a high-burden setting

“…By means of Sanger sequencing and BLAST searches, NTM culture strains were evaluated for identification using the molecular markers 16S, rpoB, and hsp65 genes. These three markers have been widely used to identify mycobacteria (45)(46)(47)(48)(49). The PCR amplification of the three genes was performed using GoTaq ® Green Master Mix (Promega, Wisconsin, United States) following protocols described elsewhere (46,47,49).…”

“…These three markers have been widely used to identify mycobacteria (45)(46)(47)(48)(49). The PCR amplification of the three genes was performed using GoTaq ® Green Master Mix (Promega, Wisconsin, United States) following protocols described elsewhere (46,47,49). Amplification of fragments was confirmed by electrophoresis in 2% UltraPure™ Agarose (Invitrogen, California, United States) gels of 15 cm x 10 cm in 0.5X Tris-boric acid-EDTA buffer at 100 V for 3 h using a ladder 100 bp Plus Opti-DNA Marker (Cat.…”

Unveiling the Clinical Diversity in Nontuberculous Mycobacteria (NTM) Infections: A Comprehensive Review

“…Polymerase chain reaction (PCR) assays have become indispensable tools for rapidly and accurately identifying NTM species in clinical specimens. These assays target specific genes or regions of the NTM genome, such as the 16S rRNA or hsp65 genes, which are highly conserved and specific to mycobacteria [ 42 ]. PCR offers several advantages, including high sensitivity and specificity and the ability to provide rapid results.…”