Abstract
BackgroundMycobacteria have a spectrum of virulence and different susceptibilities to antibiotics. Distinguishing mycobacterial species is vital as patients with non-tuberculous mycobacterial (NTM) infections present clinical features that are similar to those of patients with tuberculosis. Thus, rapid differentiation of Mycobacterium tuberculosis complex from NTM is critical to administer appropriate treatment. Hence the aim of the study was to rapid identification of mycobacterial species present in bronchial washings using multiplex real time Polymerase Chain Reaction (PCR) and to determine the drug susceptibility in identified mycobacterial species.MethodsSputum smear negative bronchoscopy specimens (n = 150) were collected for a period of one year, from patients attending the General Hospital Kandy, Sri Lanka. The specimens were processed with modified Petroff’s method and were cultured on Löwenstein– Jensen medium. DNA, extracted from the mycobacterial isolates were subjected to a SYBR green mediated real time multiplex, PCR assay with primers specific for the M. tuberculosis complex, M. avium complex, M. chelonae-M.abscessus group and M. fortuitum group. DNA sequencing was performed for the species confirmation, by targeting the 16S rRNA gene and the drug susceptibility testing was performed for the molecularly identified isolates of M. tuberculosis and NTM.ResultsThe optimized SYBR Green mediated multiplex real-time PCR assay was able to identify the presence of genus Mycobacterium in 25 out of 26 AFB positive isolates, two M. tuberculosis complex, three M. avium complex and two isolates belonging to M. chelonae-M. abscessus group. DNA sequencing confirmed the presence of M. tuberculosis, M. chelonae-M. abscessus, M. intracellulare, M. avium, Rhodococcus sp. and M. celatum. Remaining isolates were identified as Mycobacterium sp. All the NTM isolates were sensitive to amikacin and seven were resistant to ciproflaxacin. Twenty two of the NTM isolates and the isolate Rhodococcus was resistant to clarithromycin. The two isolates of M. tuberculosis were sensitive to all first line anti tuberculosis drugs.ConclusionThe optimized SYBR Green mediated multiplex real time PCR assay could be an effective tool for the rapid differentiation of pathogenic M. tuberculosis complex from the opportunistic nontuberculous mycobacteria and also it confirmed the presence of NTM in 15.3 % of the study population.
Highlights
IntroductionMycobacteria have a spectrum of virulence and different susceptibilities to antibiotics
Mycobacteria have a spectrum of virulence and different susceptibilities to antibiotics and it is believed that altered target proteins produced due to specific gene alterations such as mutations, insertions or deletions are influencing the degree of susceptibility to the drug [8]
When mycobacterial culture was considered as the gold standard there were 26 isolates (17.3 %) from 23 patients belonging to the Mycobacterium genus and among theses 23 (15.3 %) were non-tuberculous mycobacterial (NTM)
Summary
Mycobacteria have a spectrum of virulence and different susceptibilities to antibiotics. Distinguishing mycobacterial species is vital as patients with non-tuberculous mycobacterial (NTM) infections present clinical features that are similar to those of patients with tuberculosis. Pulmonary infections caused by non-tuberculous mycobacteria (NTM), i.e. Mycobacterium species that are not members of MTC are in the rise [2]. Mycobacteria have a spectrum of virulence and different susceptibilities to antibiotics and it is believed that altered target proteins produced due to specific gene alterations such as mutations, insertions or deletions are influencing the degree of susceptibility to the drug [8]. The treatment of diseases with MTC and NTM are different because many of the first and second line anti-tuberculous drugs are ineffective against many NTM [2]. Incorrect diagnosis of pulmonary diseases, i.e. if priority is given only for MTC and avoiding the presence of NTM, will lead to inappropriate treatments and the patients will not respond to conventional therapy [9]
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