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.

Trial registration: ClinicalTrials.gov NCT00396084.

FIG. 1.

Decline in M. tuberculosis mRNA and CFU in sputum from patients with TB during 7 days of INH monotherapy. Sputum was collected during a 12-h period for 2 days before and then daily during 7 days of INH administration. (Time zero is the mean of the two samples prior to INH.) Data are mean values in molecules of mRNA or CFU/ml (log₁₀) of sputum ± standard deviation for each time interval. icl, isocitrate lyase; rrnA-P1, noncoding ribosomal promoter region; hspX, alpha-crystalline protein; fbpB, fibronectin-binding protein, antigen 85B.

FIG. 2.

Decline in icl mRNA levels in sputum from patients with TB receiving INH monotherapy for 7 days compared with the decline in icl mRNA in sputum from patients receiving fluoroquinolone monotherapy (results combined for the three groups on moxifloxacin, gatifloxacin, and levofloxacin). Data are mean values in molecules of mRNA/ml (log₁₀) of sputum ± standard deviation for each time interval. FQ, fluoroquinolones; icl, isocitrate lyase.