Plasma drug activity assay for treatment optimization in tuberculosis patients

Abstract

Low antituberculosis (TB) drug levels are common, but their clinical significance remains unclear, and methods of measurement are resource intensive. Subjects initiating treatment for sputum smear-positive pulmonary TB were enrolled from Kibong'oto National TB Hospital, Tanzania, and levels of isoniazid, rifampin, ethambutol, and pyrazinamide were measured at the time of typical peak plasma concentration (C(2 h)). To evaluate the significance of the effect of observed drug levels on Mycobacterium tuberculosis growth, a plasma TB drug activity (TDA) assay was developed using the Bactec MGIT system. Time to detection of plasma-cocultured M. tuberculosis versus time to detection of control growth was defined as a TDA ratio. TDA assays were later performed using the subject's own M. tuberculosis isolate and C(2 h) plasma from the Tanzanian cohort and compared to drug levels and clinical outcomes. Sixteen subjects with a mean age of 37.8 years ± 10.7 were enrolled. Fourteen (88%) had C(2 h) rifampin levels and 11 (69%) had isoniazid levels below 90% of the lower limit of the expected range. Plasma spiked with various concentrations of antituberculosis medications found TDA assay results to be unaffected by ethambutol or pyrazinamide. Yet with a range of isoniazid and rifampin concentrations, TDA exhibited a statistically significant correlation with drug level and drug MIC, and a TDA of ~1.0 indicated the presence of multidrug-resistant TB. In Tanzania, low (≤ 2.0) TDA was significantly associated with both lower isoniazid and rifampin C(2 h) levels, and very low (≤ 1.5) TDA corresponded to a trend toward lack of cure. Study of TDA compared to additional clinical outcomes and as a therapeutic management tool is warranted.

Fig. 1.

Comparison of anti-TB drug activities (TDA) of isoniazid and rifampin concentrations tested with the M. tuberculosis clinical isolates and laboratory strain H37Rv presented in checkerboard format. TDA assays were performed using M. tuberculosis isolates and volunteer plasma alone or plasma spiked with various concentrations of isoniazid (INH) or rifampin (RMP), including concentrations below the expected C_{2 h} range (INH, 1 μg/ml; RMP, 4 μg/ml), in the low-normal range (INH, 3 μg/ml; RMP, 8 μg/ml), and in the high-normal range (INH, 5 μg/ml; RMP, 16 μg/ml). The expected C_max range for INH was 3 to 5 μg/ml and for RMP was 8 to 24 μg/ml. TDA was reported as a ratio of time to positivity of plasma-cocultured M. tuberculosis versus time to detection of M. tuberculosis alone, where a TDA ratio of 1.0 indicates stasis, a TDA ratio of >1.0 indicates killing, and a TDA ratio of <1.0 indicates growth. All isolates were susceptible to INH and RMP according to results determined by the 1% proportions method. For the combination of INH at 1 μg/ml with RMP at 8 μg/ml and INH at 5 μg/ml with RMP at 8 μg/ml, the mean TDA levels were 2.55 ± 0.06 and 4.11 ± 0.18 for isolate 1 (P = 0.008) (panel a) and 1.86 ± 0.02 and 2.12 ± 0.06 for isolate 2 (P = 0.03) (panel b). For H37Rv, RMP concentrations of ≥ 8.0 μg/ml were sterilizing.

Fig. 2.

Comparison of TB drug activity (TDA) to isoniazid and rifampin drug concentrations and drug concentrations/MIC. TDA assays were performed using plasma spiked with isoniazid (INH) or rifampin (RMP) in isolation for clinical isolates. TDA was compared to INH concentration (a), INH concentration/MIC (b), RMP concentration (c), and RMP concentration/MIC (d). Correlation determinations were performed using the Pearson coefficient.

Fig. 3.

TB drug activity (TDA) with plasma alone compared to concentrations of isoniazid, rifampin, and ethambutol for multidrug-resistant (MDR) M. tuberculosis isolates. TB drug activity (TDA) ratios were determined for volunteer plasma without drug versus plasma spiked with a various concentrations of isoniazid (INH), rifampin (RMP), and ethambutol (EMB) within the expected C_{2 h} range. The drug MICs for MDR isolate 1 were as follows: INH, 32 μg/ml (100% resistant as determined by the proportions method); RMP, 32 μg/ml (100% resistant); EMB, 5 μg/ml (<1% susceptible). The drug MICs for MDR isolate 2 were as follows: INH, 16 μg/ml (63% resistant); RMP, 16 μg/ml (77% resistant); EMB, 10 μg/ml (27% resistant).

Fig. 4.

Use of TB drug activity (TDA) determinations in MDR-TB treatment. Plasma from subjects 1, 2, 10, and 11 under treatment for drug-susceptible TB with a regimen of isoniazid, rifampin, ethambutol, and pyrazinamide administration was tested using the TDA assay and recent MDR-TB isolates from Kibong'oto National TB Hospital in Tanzania. In addition to the initial 16 subjects with drug-susceptible TB, subject B produced MDR-TB isolate B and had a TDA assay performed while on an MDR-TB regimen of ethambutol, pyrazinamide, amikacin, levofloxacin, and cycloserine.