Fig. 1.

Calculating the VBNR subpopulation using flow cytometry data. The figure shows histogram data for Turbo FP635 fluorescence intensity measured at 0 h (red) and 120 h (orange) post THP-1 infection and for 120 h in vitro culture (dotted line). Histograms (a–d) is representative of a technical replicate for the isolate S153dx from the cured group. Histograms e-h is representative of a technical replicate for isolate S43dx from the failed/recurrent group. M. tuberculosis recovered from macrophages at 0 h post infection shows maximum red fluorescence intensity (a, e). Measured Turbo FP635 signal larger than the mean fluorescence intensity was deemed high red, as measured by the bar. This gate was applied to l20h Turbo FP635 intensities measured for M. tuberculosis recovered at 120 h post infection (c, g) and in vitro cultured M. tuberculosis (b, f). The macrophage-enriched VBNR subpopulation percentage was calculated by subtracting the percentage in vitro cultured M. tuberculosis from the percentage intracellular M. tuberculosis at 120 h. A histogram overlay for the cured sample (d) shows a complete overlap for Turbo FP635 intensity measured at 120 h post macrophage infection and in vitro cultured M. tuberculosis. This is indicative of a miniscule or absent VBNR subpopulation. The histrogram overlay for the representative failed isolate (h) shows that the fluorescence intensity signal measured at 120 h post infection does not fully overlap with that of the in vitro cultured bacteria, suggesting a high red fluorescent subpopulation of bacteria, suggestive of a VBNR population.