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...
Aims: To develop and evaluate a novel genotypic test for rapid detection of rifampicin and isoniazid resistance of multidrug‐resistant (MDR) Mycobacterium tuberculosis isolates by a multiplex probe array. Methods and Results: A multiplex probe array was designed for genotypic test to simultaneously screen the mutations of rpoB, katG, inhA and ahpC genes, associated with rifampin and isoniazid resistance in M. tuberculosis, with a probe detecting one of the recently confirmed genetic markers of isoniazid resistance ahpC‐6 and ‐9 locus added. By using the genotypic test developed, 52 MDR isolates were identified, among which 46 isolates had mutations in rpoB (88·5%) and 45 at codon 315 of katG, regulatory region of inhA and oxyR‐ahpC intergenic region (86·5%), whereas all 35 susceptible isolates identified showed a wild‐type hybridization pattern. The sensitivity and specificity were 88·5% and 100% for rifampicin resistance, and 86·5% and 100% for isoniazid resistance, respectively. Conclusion: A rapid and simultaneous detection of rifampicin and isoniazid resistance caused by the mutations of rpoB, katG, inhA and ahpC genes in M. tuberculosis isolates could be achieved by a multiplex probe array developed. Significance and Impact of the Study: This genotypic test protocol has the potential to be developed on clinical application for the rapid detection of drug resistant M. tuberculosis isolates before an efficient chemotherapy is initiated.
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