Intracellular ESAT-6 vs flow cytometric analysis of CD4+IFN-γ+ cells for the diagnosis of mycobacterium tuberculosis infection in immunosuppressed patients

Background: CD4+ cells expressing IFN-γ following stimulation with the mycobacterial antigen ESAT-6, identified with flow cytometry (FCM-CD4+IFN-γ+ESAT-6+) indicate Mycobacterium Tuberculosis (MTB) infection. Another novel method to identify MTB infection is to examine the expression of intracellular ESAT-6 (i/cESAT-6) directly in host cells, thus verifying the presence of active bacilli (Poulakis, N. et al. Cytometry B 2014; doi: 10.1002/cyto.b.21220). We assessed the expression of i/cESAT-6 in comparison to FCM-CD4+IFN-γ+ESAT-6+ in medically immunosuppressed patients under biologic treatment. Methods: We performed FCM-CD4+IFN-γ+ESAT-6+ analysis and examined i/cESAT-6 expression in the blood of 64 patients with rheumatic diseases, under treatment with anti-TNFa and other biologic agents. Results: From 31 immunosuppressed patients positive for FCM, only 20 were i/cESAT-6 positive, possibly identifying the subgroup with true infection that would benefit from chemoprophylaxis. Ten FCM negative patients were i/cESAT-6 positive, possibly identifying patients that immunosuppressive treatment impaired immune response and memory but are at increased risk for progression to active disease and may require chemoprophylaxis. The fact that 11 patients are positive for IFN-γ production and negative for the presence of i/cESAT-6 protein possibly reflects previously acquired immunity. Conclusion: Intracellular ESAT-6 detection could help refine chemoprophylaxis administration in immunosuppressed patients.