Ann Lab Med.  2014 May;34(3):203-209. 10.3343/alm.2014.34.3.203.

Evaluation of Propidium Monoazide Real-Time PCR for Early Detection of Viable Mycobacterium tuberculosis in Clinical Respiratory Specimens

• Kim YJ
• Lee SM
• Park BK
• Kim SS
• Yi J
• Kim HH
• Lee EY
• Chang CL

• KMID: 1791921
• DOI: http://doi.org/10.3343/alm.2014.34.3.203

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 DeltaC(T) values (C(T) value in PMA-treated sputum samples-C(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 C(T) value changes after PMA treatment were compared between culture-positive and culture-negative groups.
RESULTS
In MTB suspensions, the increase in the C(T) 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 DeltaC(T) 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 DeltaC(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.

Keyword

Mycobacterium tuberculosis; Propidium monoazide; Real-time PCR

MeSH Terms

Adult
Aged
Area Under Curve
Azides/*chemistry
DNA, Bacterial/*analysis
Female
Humans
Lung Diseases/diagnosis/*microbiology/pathology
Male
Middle Aged
Mycobacterium tuberculosis/genetics/*isolation & purification
Pilot Projects
Propidium/*analogs & derivatives/chemistry
ROC Curve
*Real-Time Polymerase Chain Reaction
Sputum/microbiology
Tuberculosis/*diagnosis/microbiology
Azides
DNA, Bacterial
Propidium

Figure

• 

  Fig. 1 Differences between the CT values of PMA-treated and PMA-untreated aliquots of 15 Mycobacterium tuberculosis clinical isolates. Compared with the PMA-untreated live (A) group, increases in CT values in the PMA-treated live (A) group ranged from -1.5 to 4.9 and were not significant; however, compared with the results for the PMA-untreated heat inactivated (B) group, increases in the CT values in the PMA treated heat-inactivated (B) group ranged from -0.8 to 17.7 and were significant (Wilcoxon signed rank test, P=0.107, P=0.0001, respectively).*For 1 case, a 38.9 CT value was seen in the PMA-untreated heat-inactivated (B) group, and a CT value of "not detected" was seen in the PMA-treated heat-inactivated (B) group.Abbreviation: PMA, propidium monoazide.

• 

  Fig. 2 ROC curve of ΔCT values in clinical respiratory specimens, in which AFB cultures were regarded as the reference method for determining the viability of cells. The area under the curve (AUC) was 0.936. The cutoff ΔCT value (*) for maximum sensitivity (89.9%) and specificity (85.7%) in determining live cells was 3.4.Abbreviations: AFB, acid-fast bacilli; ΔCT, CT of PMA-treated specimen-CT of PMA-untreated specimen.

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