title: Evaluation of propidium monoazide real-time PCR for early detection of viable Mycobacterium tuberculosis in clinical respiratory specimens.
abstract: BACKGROUND
  Conventional acid-fast bacilli (AFB) staining cannot differentiate viable from dead cells. Propidium monoazide (PMA) is a photoreactive DNA-binding dye that inhibits PCR amplification by DNA modification. We evaluated whether PMA real-time PCR is suitable for the early detection of viable Mycobacterium tuberculosis (MTB) in clinical respiratory specimens.
  
  
  METHODS
  A total of 15 diluted suspensions from 5 clinical MTB isolates were quadruplicated and subjected to PMA treatment and/or heat inactivation. Eighty-three AFB-positive sputum samples were also tested to compare the ΔCT values (CT value in PMA-treated sputum samples-CT value in non-PMA-treated sputum samples) between culture-positive and culture-negative specimens. Real-time PCR was performed using Anyplex MTB/NTM Real-Time Detection (Seegene, Korea), and the CT value changes after PMA treatment were compared between culture-positive and culture-negative groups.
  
  
  RESULTS
  In MTB suspensions, the increase in the CT value after PMA treatment was significant in dead cells (P=0.0001) but not in live cells (P=0.1070). In 14 culture-negative sputum samples, the median ΔCT value was 5.3 (95% confidence interval [CI], 4.1-8.2; P<0.0001), whereas that in 69 culture-positive sputum samples was 1.1 (95% CI, 0.7-2.0). In the ROC curve analysis, the cutoff ΔCT value for maximum sensitivity (89.9%) and specificity (85.7%) for differentiating dead from live cells was 3.4.
  
  
  CONCLUSIONS
  PMA real-time PCR is a useful approach for differentiating dead from live bacilli in AFB smear-positive sputum samples.
authors.affiliation: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea.
authors.forename: Young Jin
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea.
authors.forename: Sun Min
authors.initials: SM
authors.lastname: Lee
authors.affiliation: Department of Physical Medicine & Rehabilitation, Korea University College of Medicine, Seoul, Korea.
authors.forename: Byung Kyu
authors.initials: BK
authors.lastname: Park
authors.affiliation: Department of Social Studies of Medicine, Pusan National University School of Medicine, Yangsan, Korea.
authors.forename: Sung Soo
authors.initials: SS
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea.
authors.forename: Jongyoun
authors.initials: J
authors.lastname: Yi
authors.affiliation: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea.
authors.forename: Hyung Hoi
authors.initials: HH
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea.
authors.forename: Eun Yup
authors.initials: EY
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea. ; Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, Korea.
authors.forename: Chulhun Ludgerus
authors.initials: CL
authors.lastname: Chang
mesh_terms: D000328:Adult; D000368:Aged; D019540:Area Under Curve; D001386:Azides / Q000737:chemistry*; D004269:DNA, Bacterial / Q000032:analysis*; D005260:Female; D006801:Humans; D008171:Lung Diseases / Q000175:diagnosis / Q000382:microbiology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010865:Pilot Projects; D011419:Propidium / Q000031:analogs & derivatives* / Q000737:chemistry; D012372:ROC Curve; D060888:Real-Time Polymerase Chain Reaction; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D001386:Azides; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium
keywords: Mycobacterium tuberculosis; Propidium monoazide; Real-time PCR