title: Real time PCR for the rapid identification and drug susceptibility of Mycobacteria present in Bronchial washings.
abstract: BACKGROUND
  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. 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.
  
  
  METHODS
  Sputum 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The 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.
authors: National Institute of Fundamental Studies, Kandy, Sri Lanka.
authors: Thilini Piushani
authors: TP
authors: Keerthirathne
authors: National Institute of Fundamental Studies, Kandy, Sri Lanka. cellbio@ifs.ac.lk.
authors: Dhammika Nayoma
authors: DN
authors: Magana-Arachchi
authors: Respiratory Disease Treatment Unit &Teaching Hospital, Kandy, Sri Lanka.
authors: Dushantha
authors: D
authors: Madegedara
authors: Department of Molecular Biology and Biotechnology, University of Peradeniya, Peradeniya, Sri Lanka.
authors: Suneth Sithumini
authors: SS
authors: Sooriyapathirana
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D002648:Child; D017931:DNA Primers; D005260:Female; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D009170:Nontuberculous Mycobacteria / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013183:Sputum / Q000382:microbiology; D013188:Sri Lanka; D014376:Tuberculosis / Q000382:microbiology; D055815:Young Adult
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S
keywords: MAC; MCAG; Multiplex real-time PCR; NTM; SYBR green