The use of the lytic D29 mycobacteriophages for accelerated determination of the drug sensitivity of tuberculosis mycobacteria to first and second-line anti-TB drugs in clinical isolates obtained after initial cultivation

Abstract

Introduction: Accelerating and improvement of determining the drug sensitivity of tuberculosis mycobacteria (MTB) in clinical isolates is an urgent problem in the treatment of tuberculosis.

Aims and Objectives: To develop a new culture format for the study of drug sensitivity of MTB using lytic mycobacteriophage and compare the results with studies in the MGIT Bactec system.

Methods: Middlebrook 7H9 liquid nutrient medium with 1mM CaCL2 and OADC enrichment in a 24-well plate format is used. 200 μl of MTB culture, obtained after initial cultivation in the MGIT Bactec system and 800 μl of the culture medium is added to the plate wells. Antibiotics at critical concentrations were added to the plate wells with the exception of control wells (without antibiotics) and incubated for 48 hours. 50 μl of a lytic mycobacteriophage D 29 (10⁷ pfu/ml) was added to each well and incubated for another 48 hours. DNA was isolated and analyze MTB single-copy RegX gene and a specific DNA fragment of D29 using quantitative real-time PCR. The results of the determination of drug sensitivity were positive with differences in the threshold cycle of test samples and a control sample of at least three cycles.

Results: When comparing the used method and the MGIT Bactec system of 108 clinical isolates, the coincidence of the results of determining drug sensitivity was shown in 98% and 97.5% of cases, respectively.

Conclusions: The method for determining drug sensitivity using lytic mycobacteriophage was accelerated up to 5 days, simple to implement and cost-effective.