In-house bulk and single cell antigen-specific whole blood Interferon-gamma assays for surveillance of M. tuberculosis infection

Department of Infectious, Parasitic and Immune-mediated Diseases, and Centre of Epidemiology, Surveillance and Health Promotion, Istituto Superiore di Sanità, 00161, Rome, Italy; Second Division of Health Department, Translational Research Unit National Institute for Infectious Diseases ‘Lazzaro Spallanzani’, Rome, Italy

^* To whom correspondence should be addressed. Email: clara.ausiello{at}iss.it.

	Abstract

Tests based on Interferon (IFN)-gamma assay (IGA) are used as adjunctive tool for diagnosis of tuberculosis infection. Here we compare in-house and commercial whole blood IGAs to identify a suitable assay for tuberculosis surveillance in population studies. The IGAs were selected on the basis of ease to perform, requiring small amount of biological sample and not requiring cell purification. Since a gold standard for LTBI is not available, sensitivity and specificity of the IGAs were determined in clinically-diagnosed active tuberculosis patients and in BCG-unvaccinated healthy controls. The in-house tests consisted in a bulk assay based on diluted whole blood and a single cell assay based on IFN-gamma intracellular staining. Commercial assays were QuantiFERON^® -TB-Gold (Q-TB) and Q-TB in tube tests. When PPD was used as antigen, in-house whole blood intracellular staining showed highly discriminatory power between active tuberculosis patients and BCG-vaccinated healthy controls, whereas the other IGAs did not discriminate the two categories. When M. tuberculosis specific antigens were used, very strong agreement between Q-TB in tube and clinical diagnosis was observed, while Q-TB assay using manufacturer instruction showed a significantly lower performance. Intriguingly, when the test was performed using RD1 proteins instead of peptides, its sensitivity was significantly increased. The in-house diluted whole blood assay showed elevated sensitivity and specificity and agreement with clinical diagnosis, and considering that it uses 1:20 of sample with respect to commercial IGA, it appears particularly promising for pediatric studies. Overall, the different assays showed different performance that needs to be considered for surveillance of tuberculosis in population studies.