Standardized Drug Susceptibility Testing

Each laboratory followed a standard operating protocol laid out by the CRyPTIC consortium, which was similar to that described previously (17 (link)). Clinical samples were sub-cultured either using Lowenstein-Jensen tubes, 7H10 agar plates or MGIT tubes. The protocol specified that first a suspension at 0.5 McFarland standard in saline Tween with glass beads (Thermo Fisher, Scientific Inc., USA) from 20- to 25-day-old colonies. These were then diluted 100-fold by adding 100 μl of suspension to 10 ml of enriched 7H9 broth (17 (link)). A semi-automated Sensititre Autoinoculator (Thermo Fisher, Scientific Inc., USA) was used to dispense 100 μl of inoculum (1.5 × 10⁵ CFU/ml, with approximate range from 5 × 10⁴ CFU/ml to 5 × 10⁵ CFU/ml) into a well of a UKMYC5/6 microdilution plate. The plate was then sealed using transparent plastic provided by the manufacturer. The UKMYC5 and UKMYC6 microdilution plates were designed by the CRyPTIC consortium and manufactured by Thermo Fisher Inc., U.K. The drugs included and their concentrations are described in Fig. 1. Delamanid and bedaquiline pure substances were provided by Otsuka Pharmaceutical Co., Ltd. and Jannsen Pharmaceutica, respectively. The H37Rv ATCC 27294 was used to perform periodic quality control runs since it is susceptible to all the drugs on both plate designs.