Our aim was to develop a Mycobacterium tuberculosis (Mtb) growth inhibition assay (MGIA) as a summary estimate of host immune control of virulent Mtb. Mycobacterial growth inhibition (MGI) using previously frozen human PBMCs infected with H37Rv was assessed by live-cell imaging (Incucyte©) complemented by imaging flow cytometry analysis of phagocytosis. MGI measured as relative fluorescence units (RFU) was calibrated to time to positive culture (TTP) in BACTEC 960 MGIT. At a MOI (multiplicity of infection) of 5, there was a wide range of MGI of blood donors (1.1*106-2.7*106 RFU, n = 14). Intra- and inter-assay variability were at most 17.5 and 20.7 CV%. Cell viability at day 5 was 57 and 62% monitored by the LDH and Draq7 assays respectively. There was a strong correlation between a readout for Mtb growth using CFU counts or TTP compared to RFU (r2≥0.96). Our MGIA enabling live-cell imaging and monitoring of cell viability was able to detect a wide range of Mtb growth inhibition by PBMCs and was calibrated to several readout options for bacterial growth. This MGIA may be valuable as a surrogate marker of host immunity in a personalized medicine approach.

中文翻译：

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一种新型分枝杆菌生长抑制试验，采用强毒结核分枝杆菌的活细胞成像和宿主细胞活力监测

我们的目标是开发一种结核分枝杆菌 (Mtb) 生长抑制试验 (MGIA)，作为对强毒 Mtb 的宿主免疫控制的总结估计。使用感染 H37Rv 的先前冷冻的人 PBMC 的分枝杆菌生长抑制 (MGI) 通过活细胞成像 (Incucyte©) 评估，辅以吞噬作用的成像流式细胞术分析。以相对荧光单位 (RFU) 测量的 MGI 在 BACTEC 960 MGIT 中校准到阳性培养时间 (TTP)。在 MOI（感染复数）为 5 时，献血者的 MGI 范围很广（1.1*106-2.7*106 RFU，n = 14）。测定内和测定间变异性最多为 17.5 和 20.7 CV%。通过 LDH 和 Draq7 检测，第 5 天的细胞活力分别为 57% 和 62%。与 RFU (r2≥0.96) 相比，使用 CFU 计数或 TTP 的 Mtb 生长读数之间存在很强的相关性。我们的 MGIA 能够实现活细胞成像和细胞活力监测，能够检测到 PBMC 对 Mtb 生长的广泛抑制，并针对细菌生长的多个读数选项进行了校准。这种 MGIA 可能作为个性化医疗方法中宿主免疫的替代标志物很有价值。