Evaluation of propidium monoazide real-time PCR for early detection of viable Mycobacterium tuberculosis in clinical respiratory specimens.
Young Jin Kim, Sun Min Lee, Byung Kyu Park, Sung Soo Kim, Jongyoun Yi, Hyung Hoi Kim, Eun Yup Lee, Chulhun Ludgerus Chang
Author Information
Young Jin Kim: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea.
Sun Min Lee: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea.
Byung Kyu Park: Department of Physical Medicine & Rehabilitation, Korea University College of Medicine, Seoul, Korea.
Sung Soo Kim: Department of Social Studies of Medicine, Pusan National University School of Medicine, Yangsan, Korea.
Jongyoun Yi: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea.
Hyung Hoi Kim: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea.
Eun Yup Lee: Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea.
Chulhun Ludgerus Chang: 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.
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.
