propidium-monoazide and Tuberculosis

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

Topics: Adult; Aged; Area Under Curve; Azides; DNA, Bacterial; Female; Humans; Lung Diseases; Male; Middle Aged; Mycobacterium tuberculosis; Pilot Projects; Propidium; Real-Time Polymerase Chain Reaction; ROC Curve; Sputum; Tuberculosis

The slow turnaround time for Mycobacterium tuberculosis drug susceptibility results is a barrier to care. We developed a rapid quantitative PCR (qPCR)-based phenotypic antimicrobial susceptibility test that utilizes amplification of the M. tuberculosis 16S rRNA gene after 3 days of incubation with antituberculosis drugs. To decrease background from killed organisms, we used propidium monoazide (PMA), a DNA-binding dye that penetrates damaged bacterial cells and renders DNA unamplifiable. M. tuberculosis was cultured in broth media containing PMA with or without drugs for 3 days prior to DNA extraction and real-time PCR amplification. 16S rRNA qPCR exhibited a significant decrease in threshold cycle (C(T)) time values (C(T) control - C(T) drug treated) with drug-susceptible strains compared with resistant strains. Susceptibility data were reported as ΔCT or as 2(Δ)(CT) and with appropriate cutoffs yielded an accuracy of 89 to 100% on 38 susceptible, multidrug-resistant, and extensively drug-resistant strains compared with conventional agar proportion susceptibility results for isoniazid, rifampin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide, para-aminosalicylic acid, linezolid, and cycloserine and compared with Bactec MGIT results for pyrazinamide. This PMA-qPCR assay is useful as a rapid 3-day first- and second-line drug susceptibility test for M. tuberculosis.

Topics: Antitubercular Agents; Azides; Enzyme Inhibitors; Humans; Microbial Sensitivity Tests; Microbial Viability; Mycobacterium tuberculosis; Polymerase Chain Reaction; Propidium; RNA, Bacterial; RNA, Ribosomal, 16S; Time Factors; Tuberculosis