Sputum Mycobacterium tuberculosis mRNA as a Marker of Bacteriologic Clearance in Response to Antituberculosis Therapy

Abstract

mRNA is a marker of cell viability. Quantifying Mycobacterium tuberculosis mRNA in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mRNA levels were measured in sputum specimens from patients with tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (IL-2) trial. In the early bactericidal activity study, sputum for quantitative culture and mRNA analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, Bactec 460 liquid culture, and mRNA analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-PCR assays for several gene targets. mRNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mRNA correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mRNA is a reliable marker of M. tuberculosis viability.The development of new drugs for tuberculosis (TB) treatment has been hampered by the lack of an early surrogate marker that reflects nonrelapsing cure. Two-month sputum culture conversion on solid medium is the best-established predictor of treatment outcome (12). Early bactericidal activity (EBA), measured as the decline in sputum Mycobacterium tuberculosis colony counts (CFU) during treatment, is a commonly used tool for comparing new drugs to current drugs and dose finding, but quantitative culture is time-consuming and labor-intensive (4). An ideal marker would measure events early during treatment and be accurate regardless of the mechanism of drug action or the regimen being tested. In addition to serial sputum culture conversion, less well studied biomarkers of response to therapy include sputum 85B protein and mRNA (2, 17), sputum cytokines (15), and whole-blood bactericidal activity (18). None has been adequately validated to demonstrate long-term efficacy in phase 3 trials.In prior work we demonstrated that levels of M. tuberculosis fbpB mRNA (encoding fibronectin-binding protein, antigen 85B) and hspX (encoding alpha-crystalline homologue protein) mRNA decline rapidly in conjunction with M. tuberculosis colony counts after initiation of a rifampin-based standard drug therapy (2) and in a 14-day EBA study of rifalazil where both isoniazid and a rifamycin were given simultaneously (unpublished data). In most cases the solidmedium culture was still positive...

Molecular Bacterial Load Assay, a Culture-Free Biomarker for Rapid and Accurate Quantification of Sputum Mycobacterium tuberculosis Bacillary Load during Treatment

“…Previous reports have suggested that abundant mRNA species such as fbpB-85B and icl may be useful for the detection of live bacilli (5,9,10,15). While the clearance of the icl gene showed promise as a biomarker for a fall in CFU at 7 days, it did not match liquid culture sensitivity at day 56.…”

The early bactericidal activity of antituberculosis drugs

“…These include the PCR [12,42], antigen 85 induction [69,70], whole blood bactericidal assay [71,72], the Early bactericidal activity of antituberculosis drugs Review www.expert-reviews.com measurement of mRNA [73,74], molecular bacterial load assay [75] and most recently the use of GeneXpert MTB/RIF to quantitate mycobacteria in sputum [76,77]. Each of these techniques could potentially either add value to the current determination of the fall in sputum CFU counts or replace it.…”

Persistence of DNA in a Cured Patient and Positive Culture in Cases with Low Antibody Levels Bring into Question Diagnosis of Q Fever Endocarditis

“…This innovative concept must be confirmed in a future study. Recently, RNA detection with RT-PCR has also been used to determine bacterial viability in other infectious diseases (26)(27)(28)(29).…”