Abstract
M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the MICs to be elucidated. The three participating laboratories each inoculated 38 96-well plates with 15 known M. tuberculosis strains (including the standard H37Rv reference strain) and, after 2 weeks' incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. The AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (>30000) of samples of M. tuberculosis from patients over the next few years.
Highlights
Existing liquid and solid media culture-based DST methods require significant infrastructure and highly-trained laboratory scientists and, due to the inherent slow-growth rate of M. tuberculosis, take at least 4-5 weeks to return a result to the clinician [4]
After two weeks of incubation, the minimum inhibitory concentrations (MICs) of the 14 anti-TB drugs on each plate were read by a trained laboratory scientist using a VizionTM instrument, a photograph of the plate was taken which was analysed at a later date by the Automated Mycobacterial Growth Detection Algorithm (AMyGDA) software
20 UKMYC5 plates were inoculated and incubated. Both AMyGDA and the laboratory scientist detected that M. tuberculosis appeared not to be growing in one of the two positive control wells of one plate: this plate was excluded from analysis
Summary
Existing liquid and solid media culture-based DST methods require significant infrastructure and highly-trained laboratory scientists and, due to the inherent slow-growth rate of M. tuberculosis, take at least 4-5 weeks to return a result to the clinician [4] Genotypic assays such as the Cepheid Xpert MTB/RIF [5] have been developed for M. tuberculosis (MTB) that overcome some of these challenges, no single molecular test exists that determines the effectiveness of a large number of anti-TB compounds simultaneously. Using whole-genome sequencing (WGS) in this way has been shown to be much faster, but is already cheaper than the existing culture-based methods [4] It critically depends, on a comprehensive and accurate catalogue that relates genetic variants to their effect on different anti-TB compounds. We intend deploying AMyGDA within the international CRyPTIC tuberculosis project to consistently determine MICs for the 14 anti-compounds on the UKUMYC5 plate for all >30,000 samples collected [10]
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