title: Polymerase chain reaction - restriction fragment length polymorphism analysis for the differentiation of mycobacterial species in bronchial washings.
abstract: OBJECTIVE
  To identify mycobacterial species in bronchoscopy specimens with a simple assay based on polymerase chain reaction and restriction enzyme digestion.
  
  
  METHODS
  Sputum smear negative, bronchoscopy specimens (n=202) were collected from patients attending the Central Chest Clinic and the Teaching Hospital Kandy, Sri Lanka. DNA, extracted from the mycobacterial cultures (n=43) were amplified using known mycobacterial specific Sp1 and Sp2 primers. Resulting products were digested with HaeIII and CfoI restriction enzymes and DNA sequencing was performed for the selected isolates.
  
  
  RESULTS
  Among the culture positive patients, PCR was able to distinguish 12 rapid growers (~280-320 bp), 15 slow (~200-220 bp) and 10 patients having both rapid and slow and one having two rapid growing mycobacteria. DNA Sequence analysis revealed the presence of M. intracellulare (n=3), M. phocaicum (n=7), M. tuberculosis complex (n=13), Nocardia (n=2), M. smegmatis (n=1) and Mycobacterium sp (n=12). The identified organisms got digested upon exposure to HaeIII restriction enzyme whereas when exposed to CfoI, only M. phocaicum yielded 80 bp and 230 bp DNA fragments while others remained undigested. Consequently, six patients were confirmed to have M. tuberculosis complex, seven had both M. tuberculosis and non-tuberculosis bacteria (NTM) in their bronchoscopy specimens while 21 had NTM.
  
  
  CONCLUSIONS
  Optimised PCR-RFLP assay was able to differentiate M. tuberculosis complex bacteria from nontuberculosis mycobacteria and Nocardia. Molecular analysis confirmed the presence of NTM in bronchoscopy specimens and according to the study a significant proportion of patients (13% to 14%) of the study population were found to have NTM in their bronchial washings.
authors.affiliation: Cell Biology, Institute of Fundamental Studies, Hantana Road, Kandy.nayomam@yahoo.com.
authors.forename: D K
authors.initials: DK
authors.lastname: Weerasekera
authors.forename: D N
authors.initials: DN
authors.lastname: Magana-Arachchi
authors.forename: D
authors.initials: D
authors.lastname: Madegedara
authors.forename: N
authors.initials: N
authors.lastname: Dissanayake
mesh_terms: D015373:Bacterial Typing Techniques; D001999:Bronchoscopy; D005260:Female; D006801:Humans; D008297:Male; D009159:Mycobacteriaceae / Q000145:classification* / Q000302:isolation & purification*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D013188:Sri Lanka