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

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 C T 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 ΔC T 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.

Original languageEnglish
Pages (from-to)203-209
Number of pages7
JournalAnnals of Laboratory Medicine
Volume34
Issue number3
DOIs
Publication statusPublished - 2014 Jan 1

Fingerprint

Mycobacterium tuberculosis
Sputum
Real-Time Polymerase Chain Reaction
Bacilli
Bacillus
ROC Curve
Acids
Suspensions
Confidence Intervals
DNA
Korea
propidium monoazide
Amplification
Coloring Agents
Hot Temperature
Staining and Labeling
Sensitivity and Specificity
Polymerase Chain Reaction

Keywords

  • Mycobacterium tuberculosis
  • Propidium monoazide
  • Real-time PCR

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Evaluation of propidium monoazide real-time PCR for early detection of viable mycobacterium tuberculosis in clinical respiratory specimens. / Kim, Young Jin; Lee, Sun Min; Park, Byung Kyu; Kim, Sung Soo; Yi, Jongyoun; Kim, Hyung Hoi; Lee, Eun Yup; Chang, Chulhun Ludgerus.

In: Annals of Laboratory Medicine, Vol. 34, No. 3, 01.01.2014, p. 203-209.

Research output: Contribution to journalArticle

Kim, Young Jin ; Lee, Sun Min ; Park, Byung Kyu ; Kim, Sung Soo ; Yi, Jongyoun ; Kim, Hyung Hoi ; Lee, Eun Yup ; Chang, Chulhun Ludgerus. / Evaluation of propidium monoazide real-time PCR for early detection of viable mycobacterium tuberculosis in clinical respiratory specimens. In: Annals of Laboratory Medicine. 2014 ; Vol. 34, No. 3. pp. 203-209.
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AU - Yi, Jongyoun

AU - Kim, Hyung Hoi

AU - Lee, Eun Yup

AU - Chang, Chulhun Ludgerus

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AB - 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 C T 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 ΔC T 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.

KW - Mycobacterium tuberculosis

KW - Propidium monoazide

KW - Real-time PCR

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