Evaluation of Propidium Monoazide Real-Time PCR for Early Detection of Viable Mycobacterium tuberculosis in Clinical Respiratory Specimens
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초록
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 suit- able for the early detection of viable Mycobacterium tuberculosis (MTB) in clinical respira- tory specimens. Methods: A total of 15 diluted suspensions from 5 clinical MTB isolates were quadrupli- cated and subjected to PMA treatment and/or heat inactivation. Eighty-three AFB-positive sputum samples were also tested to compare the ΔCγ T values ( Cγ T value in PMA-treated sputum samples− Cγ T 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 CγT value changes after PMA treatment were compared between culture-positive and culture-negative groups. Results: In MTB suspensions, the increase in the CγT value after PMA treatment was sig- nificant in dead cells ( P =0.0001) but not in live cells ( P =0.1070). In 14 culture-negative sputum samples, the median ΔCγT 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 ΔCγT 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.
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 suit- able for the early detection of viable Mycobacterium tuberculosis (MTB) in clinical respira- tory specimens. Methods: A total of 15 diluted suspensions from 5 clinical MTB isolates were quadrupli- cated and subjected to PMA treatment and/or heat inactivation. Eighty-three AFB-positive sputum samples were also tested to compare the ΔCγ T values ( Cγ T value in PMA-treated sputum samples− Cγ T 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 CγT value changes after PMA treatment were compared between culture-positive and culture-negative groups. Results: In MTB suspensions, the increase in the CγT value after PMA treatment was sig- nificant in dead cells ( P =0.0001) but not in live cells ( P =0.1070). In 14 culture-negative sputum samples, the median ΔCγT 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 ΔCγT 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.
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