Multi-parameter flow cytometry immunophenotyping distinguishes different stages of tuberculosis infection.

Objectives: To identify new potential host biomarkers in blood to discriminate between active TB patients, uninfected (NoTBI) and latently infected contacts (LTBI).

Methods: A blood cell count was performed to study parent leukocyte populations. Peripheral blood mononuclear cells (PBMCs) were isolated and a multi-parameter flow cytometry assay was conducted to study the distribution of basal and Mycobacterium tuberculosis (Mtb)-stimulated lymphocytes. Differences between groups and the area under the ROC curve (AUC) were investigated to assess the diagnostic accuracy.

Results: Active TB patients presented higher Monocyte-to-lymphocyte and Neutrophil-to-lymphocyte ratios than LTBI and NoTBI contacts (p<0.0001; AUC>0.8). Lymphocyte subsets with differences (p >0.05; AUC >0.7) between active TB and both contact groups include the basal distribution of Th1/Th2 ratio, Th1-Th17, CD4 Central Memory (T) or MAIT cells. Expression of CD154 is increased in Mtb-activated CD4 T and Effector Memory T cells in active TB and LTBI compared to NoTBI. In CD4T cells, expression of CD154 showed a higher accuracy than IFNγ to discriminate Mtb-specific activation.

Conclusions: We identified different cell subsets with potential use in tuberculosis diagnosis. Among them, distribution of CD4 T cells and their expression of CD154 after Mtb-activation are the most promising candidates.