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

Abstract

BackgroundConventional 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.MethodsA 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.ResultsIn 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.ConclusionsPMA real-time PCR is a useful approach for differentiating dead from live bacilli in AFB smear-positive sputum samples.

Selective Detection of DNA from Viable <i>Mycobacterium tuberculosis</i> Complex Strains Using the EMA-PCR Method

“…It has been reported that PMA is more suitable than EMA for the discrimination between viable and dead bacteria in NAT because EMA influences viable bacteria more than PMA (3,5). However, our experiment revealed that EMA-PCR can discriminate dead MTC cells from viable cells as accurately as the PMA-PCR method, with high specificity.…”

“…Secondly, we may be able to discriminate paradoxical results from microbiological treatment failure when clinical samples are smearpositive and PCR-positive for patients receiving tuberculosis treatment. This method can be extended to the evaluation of drug susceptibility by using differences in C T values of real-time PCR before and after EMA treatment (3)(4)(5). EMA-PCR utilizes the ability of EMA : Smear grades described according to the World Health Organization guidelines for AFB smear positivity.…”

“…However, it cannot discriminate viable from dead cells (2). To resolve this problem, the efficacy of discrimination of viable from dead bacteria using propidium monoazide (PMA) or ethidium monoazide (EMA) has been examined, leading to the development of PMA-PCR and EMA-PCR as viable bacteria-selective detection methods using PCR and/or real-time PCR (3)(4)(5). These methods enable the selective detection of viable cell-derived DNA, using membranepermeable dyes (PMA or EMA) that modify dead cell-derived DNA, thereby preventing their PCR amplification (4,6,7).…”

Assessment of treatment response in tuberculosis

“…The utility of using tests such as GeneXpert® MTB/RIF (Cepheid Sunnyvale, CA, USA) and GenoType MTBDR plus /MTBDR sl (Hain Lifescience Nehren, Germany) to ascertain sputum sterilization at any point during treatment is limited by DNA amplification from dead bacteria in clinical samples [30]. There is ongoing research to improve specificity of DNA amplification from live bacteria through treatment of sputa with agents such as propidium monazide, a DNA-binding dye that penetrates through damaged cell walls and inhibits PCR amplification via DNA modification [31]. An alternative approach includes quantification of abundant ribonucleic acid (RNA) species extracted from sputum as surrogate markers of bacterial clearance [32,33].…”

Meta-narrative review of molecular methods for diagnosis and monitoring of multidrug-resistant tuberculosis treatment in adults

“…PMA selectively intercalates the dead MTBC DNA and inhibits its amplification and detection. [61] Monitoring anti-TB treatment by Xpert-PMA has been evaluated in two studies. The first study measured bacterial load from 1937 sputum samples that were collected before treatment, 2 weeks after treatment, and monthly thereafter, during the intensive and continuation phases of non-MDR-TB and MDR-TB treatment.…”