Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy

Effective treatment regimens for pulmonary tuberculosis are difficult to as sess because of the slow growth rate of Mycobacterium tuberculosis in cultu re and its protracted clearance from sputum. A rapid method that reflects e ffective antimicrobial activity would markedly advance evaluation of treatm ent and promote the assessment of new antituberculosis drugs. Conventional methods measure the progressive reduction of numbers of acid-fast bacilli i n the sputum smear and the clearance of organisms in sputum culture. In thi s study, we measured levels of M. tuberculosis 85B (alpha antigen) messenge r RNA (mRNA), 16S ribosomal RNA (rRNA), and IS6110 DNA in patients' sputa t o ascertain whether they could serve as potential surrogate markers of resp onse to chemotherapy. Sputum specimens were sequentially collected for up t o a year from 19 smear-positive pulmonary tuberculosis patients receiving a n optimal drug treatment regimen. Nucleic acids were isolated from these sp ecimens, and two M. tuberculosis molecular targets (mRNA, DNA) were quantif ied, using the ABI Prism 7700 Sequence Detection System. The Mycobacterium genus-specific 165 rRNA was quantified with a limiting dilution RT-PCR assa y. Results show that levels of 85B mRNA declined after initiation of therap y, as did viable M. tuberculosis colony counts, with 90% of patients becomi ng negative for both markers after 2 mo of treatment. The rapid disappearan ce of M. tuberculosis mRNA from sputum suggests that it is a good indicator of microbial viability and a useful marker for rapid assessment of respons e to chemotherapy.