Background:
Tuberculosis (TB) is a massive global health problem, the leading cause of death from infection worldwide. Currently, the gold standard for detection of latent TB is the Tuberculin Skin Test (TST/Mantoux test) and detection of interferon-gamma produced by CD4+ T lymphocytes exposed to TB antigens in vitro. Both have limitations.We are developing an in vitro Diagnostic (IVD) certified, TB-specific version of the Act-T4 Cell™ kit, that robustly gives clear cut positive and negative discrimination of latent TB infection via Flow Cytometry, decreasing the need for repeat testing and subjective clinical interpretation.
Methods:
Sodium heparin anti-coagulated whole blood from known positive TB patients was incubated with pools of peptides (15mer) representing TB-specific antigens (ESAT-6, CFP-10) for 44-48hours and measured co-expression of CD25 and CD134 (OX40) on CD4+ T cells via 4-colour flow cytometry. The formulation of the TB peptides was optimized to maximize their ease of use, especially with respect to peptide solubility into whole blood. Incubation time, transport conditions and peptide stability were optimized.
Results:
Our data shows that pooled peptides can be directly diluted into culture media to give clear positive results on the OX40 assay, equivalent to those dissolved in minimal concentrations of DMSO, with optimal concentration of 10ug per peptide/test. Assay set up can be delayed up to 24hrs (4.91% CD25+/CD134+) while yielding a similar result to that set up ≤ 6hrs after taking blood (4.82% CD25+/CD134+). Incubation in a water bath or oven with CO2 Independent Media instead of Iscove’s Modified Dulbecco’s Medium in a standard 37°C CO2 Incubator does not diminish results: 5.9% versus 5.84% CD25+/CD134+ respectively.
Conclusion:
We show that our flow-based kit is a simple robust assay that may facilitate the diagnosis of TB in developing countries. This potentially has a global impact especially in countries where HIV/TB co-infection is prevalent.