C-203. Monitoring Therapy Efficacy by Real-Time Mycobacterium tuberculosis mRNA Detection in Sputum Specimens

N. Mdivani1, H. Li2, M. Akhalaia3, M. Gegia1, L. Goginashvili3, D. Kernodle2, G. Khechinashvili1, Y-W. Tang2;
1Georgian Fndn. against Tuberculosis and Lung Diseases, Tbilisi, GEORGIA, 2Vanderbilt Univ. Med. Ctr., Nashville, TN, 3Natl. Ctr. of Tuberculosis and Lung Diseases, Tbilisi, GEORGIA.

Current laboratory methods for monitoring tuberculosis (TB) therapy efficacy are based on mycobacterial culture, and a rapid, sensitive method that reflects effective anti-TB drug activity is extremely desirable. We have developed two real-time PCR TaqMan assays to target DNA of the IS6100 insertion element and mRNA of the 85B protein gene, respectively. The test performance for anti-TB therapy efficacy monitoring was validated against mycobacterial culture results obtained from sputum. A total of 65 confirmed new tuberculosis patients, who have never taken TB drugs, or have not taken for more than one month, were recruited in a prospective study in Tbilisi, Georgia. Patients were treated with isoniazid, rifampin, streptomycin and ethambutol, and followed up at two weeks, one, two, and four months after the initiation of anti-TB therapy. Sputum specimens were collected before treatment and at each follow-up visit, total nucleic acids were extracted from NaOH-NALC-decontaminated and concentrated specimens using a bioMerieux NucliSens easyMAG system and tested by the PCR TaqMan assays for mRNA and DNA. In comparison to culture, the mRNA RT-PCR assay had a sensitivity of 85.2% and a specificity of 88.6%, with an overall agreement rate of 87.1% between culture and RT-PCR among all 286 sputum specimens. In monitoring therapy efficacy, M. tuberculosis was detected by culture and mRNA RT-PCR in 65 (100.0%) and 62 (95.4%) at the time of treatment, 44 (67.7%) and 36 (55.4%) at 2 weeks, 17 (27.0%) and 18 (27.7%) at one months, 1 (1.8%) and 8 (14.3%) at two months, and 1 (2.7%) and 3 (8.1%) at four months after therapy, respectively. The mRNA RT-PCR assay agreed with culture for all follow-up time points in 36 (55.4%) cases, and predicted therapy effectiveness earlier in 13 (20.0%) and later in 13 (20.0%) cases than in culture. A significantly high false positive rate was noticed in the late follow-up stage when the DNA PCR assay was used for therapy monitoring. Our data indicate that the mRNA RT-PCR assay provides a rapid and real-time tool for anti-TB therapy efficacy monitoring.