Real time PCR for the rapid identification and drug susceptibility of Mycobacteria present in Bronchial washings

BMC Infectious Diseases

Research Article

Suneth Sithumini Sooriyapathirana¹  Dhammika Nayoma Magana-Arachchi²  Thilini Piushani Keerthirathne²  Dushantha Madegedara³ 

[1] Department of Molecular Biology and Biotechnology, University of Peradeniya, Peradeniya, Sri Lanka;National Institute of Fundamental Studies, Kandy, Sri Lanka;Respiratory Disease Treatment Unit &Teaching Hospital, Kandy, Sri Lanka;

关键词: Multiplex real-time PCR;    MCAG;    MAC;    NTM;    SYBR green;   

DOI  :  10.1186/s12879-016-1943-y

 received in 2016-01-07, accepted in 2016-10-19,  发布年份 2016

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© The Author(s). 2016