Whole Blood Mycobacterial Growth Inhibition Assay

This assay was performed as previously described^{19 (link)}. Duplicate tubes containing 300 μl of whole blood were incubated with 300 μl of RPMI (containing 10% pooled human serum, 2mM L-glutamine and 25 mM HEPES; Sigma, UK) inoculated with ~150 cfu BCG or M.tb on a 360° rotator at 37 °C for 96 hours (volume of the mycobacterial stock was calculated to give a TTP of 6.5 days, previously determined to give optimal differential responses). Cells were then lysed with sterile water, the mycobacteria resuspended in 7H9 media and transferred to a BACTEC MGIT tube supplemented with PANTA antibiotics and OADC enrichment broth (Becton Dickinson, UK). Tubes were placed in the BACTEC 960 machine and incubated at 37 °C until the detection of positivity by fluorescence (TTP). In addition, on day 0, duplicate viability control tubes were set up by directly inoculating supplemented BACTEC MGIT tubes with the same volume of mycobacteria as the samples. The mean TTP for duplicates was converted to a cfu count (as described above) and net growth ratio was calculated as Log₁₀(sample cfu/control cfu). A smaller net growth value indicates less bacillary replication and therefore represents greater mycobacterial control. Samples failing to meet pre-defined reproducibility criteria of ΔTTP < 6 hours between duplicates, as determined by initial experiments, were excluded.