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