propidium-monoazide and Tuberculosis--Pulmonary

Propidium monoazide (PMA) penetrates non-viable cells with compromised membranes. PMA has been proposed to improve the specificity of Xpert MTB/RIF (Xpert) for the detection of viable Mycobacterium tuberculosis. This study assessed the effect of PMA on Xpert cycle thresholds (C

Topics: Adolescent; Adult; Aged; Antitubercular Agents; Azides; DNA, Bacterial; Humans; Microbial Sensitivity Tests; Microbial Viability; Middle Aged; Mycobacterium tuberculosis; Propidium; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Specimen Handling; Sputum; Tuberculosis, Pulmonary; Young Adult

Reliable laboratory diagnosis of tuberculosis (TB), including laboratory biomarkers of cure, remains a challenge. In our study we evaluated the performance of a Propidium Monoazide (PMA) assay for the detection of viable TB bacilli in sputum specimens during anti-TB chemotherapy and its potential use as a TB biomarker. The study was conducted at three centres on 1937 sputum specimens from 310 adult bacteriologically confirmed pulmonary TB patients obtained before commencing anti-TB treatment and at regular intervals afterwards. Performance of the PMA assay was assessed using various readout assays with bacteriology culture results and time to positivity on liquid media used as reference standards. Treatment of sputum with N-acetyl-cysteine was found to be fully compatible with the PMA assay. Good sensitivity and specificity (97.5% and 70.7-80.0%) for detection of live TB bacilli was achieved using the Xpert(®) MTB/RIF test as a readout assay. Tentative Ct and ΔCt thresholds for the Xpert(®) MTB/RIF system were proposed. Good correlation (r = 0.61) between Ct values and time to positivity of TB cultures on liquid media was demonstrated. The PMA method has potential in monitoring bacterial load in sputum specimens and so may have a role as a biomarker of cure in TB treatment.

Topics: Adult; Antitubercular Agents; Azides; Bacterial Load; Drug Monitoring; Humans; Microbial Viability; Mycobacterium tuberculosis; Propidium; Specimen Handling; Sputum; Tuberculosis, Pulmonary; Young Adult

Diagnostic methods of TB, nowadays, are prone to delay in diagnosis, increased false negative results and are not sensitive to many forms of paucibacillary disease. The aims of this study were to implement a quantitative nucleic acid-based diagnostic test for paucibacillary tuberculosis, enabling the identification and quantification of viable Mycobacterium tuberculosis bacilli by quantitative Real-Time PCR (qRT-PCR). The intergenic region of the single-copy inhA-mabA gene was chosen as the target region for design of primers and probes conjugated with fluorophores. The construction of synthetic DNA flanking the target region served as standards for absolute quantification of nucleic acids. Using the intercaling dye, propidium monoazide, we were able to discriminate between viable and dead cells of M. tuberculosis. The diagnosis method showed a broad sensitivity (96.1%) when only compared to samples of smear-positive sputum and ROC analyses shows that our approach performed well and yielded a specificity of 84.6% and a sensitivity of 84.6% when compared to M. tuberculosis colony-forming units counting.

Topics: Affinity Labels; Azides; Colony Count, Microbial; Coloring Agents; DNA, Bacterial; DNA, Intergenic; Dose-Response Relationship, Drug; Humans; Microbial Viability; Mycobacterium tuberculosis; Propidium; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Sputum; Tuberculosis, Pulmonary

Topics: Antitubercular Agents; Azides; Bacterial Load; DNA, Bacterial; Humans; Monitoring, Physiologic; Mycobacterium tuberculosis; Polymerase Chain Reaction; Propidium; Sputum; Treatment Outcome; Tuberculosis, Pulmonary