Rapid drug susceptibility test of Mycobacterium tuberculosis using microscopic time-lapse imaging in an agarose matrix

Abstract

Tuberculosis (TB) is a major global health problem, and multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) are spreading throughout the world. However, conventional drug susceptibility test (DST) methods, which rely on the detection of the colony formation on a solid medium, require 1-2 months to the result. A rapid and accurate DST is necessary to identify patients with drug-resistant TB and treat them with appropriate drugs. Here, we used microscopic imaging of Mycobacterium tuberculosis (MTB) immobilized in an agarose matrix for a rapid DST. The agarose matrix, which was molded in a microfluidic chip, was inoculated with MTB, and TB drugs in liquid culture medium diffused throughout the agarose to reach the MTB immobilized in the agarose matrix. After the responses of MTB to drugs were tracked with an automated microscopic system, an image-processing program automatically determined the susceptibility and resistance of MTB to specific doses of TB drugs. The automatic DST system was able to assess the drug susceptibility of various drug-resistant clinical TB strains within 9 days with an accuracy comparable to that of conventional method. Our rapid DST method based on microscopic time-lapse imaging greatly reduces the time required for a DST and can be used to rapidly and accurately treat TB patients.

Bedaquiline susceptibility test for totally drug-resistant tuberculosis Mycobacterium tuberculosis

“…In this study, we employed a DAC system that uses single bacterial colony time-lapse imaging that also reduces MIC assay time. Choi et al (2016) previously applied the DAC system for rapid DST of M. tuberculosis. They showed that the DAC system provided drug susceptibility values within 9 days with an accuracy comparable to that of gold standard methods.…”

“…They showed that the DAC system provided drug susceptibility values within 9 days with an accuracy comparable to that of gold standard methods. The DAC system can provide DST with both speed and accuracy (Choi et al, 2016). Figure 1A shows DAC system determinations of bedaquiline MIC values for those TDR re- sistant M. tuberculosis strains that grew in the agarose.…”

“…This method provides rapid and accurate drug susceptibility (DST) values that coincide with the recommended performance values from the FDA (Choi et al, 2016). Furthermore, disc agarose channel (DAC) single-cell morphological analysis system was able to accurately assess the susceptibility of various TB strains within 9 days (Choi et al, 2016).…”

“…This DST method of the DAC system was previously described (Choi et al, 2016). For the DAC system, colonies grown in Löwenstein-Jensen (LJ) medium were collected using a sterilized loop.…”

“…The cell growth was automatically imaged with a 40 × lens on an inverted microscope every other day using the time-lapse method. Growth images were then automatically processed (Choi et al, 2016). The DAC drug susceptibility was determined by calculating the area of M. tuberculosis cells in the time-lapse images.…”