Advances in the development of an in vitro test for detection of tuberculosis infection

NICOLÁS O. AMIANO; NANCY TATEOSIAN; RODRIGO HERNÁNDEZ DEL PINO; ALBERTO LEVI; MARIA PAULA MORELLI; JOAQUIN PELLEGRINI; EDUARDO CHULUYAN; FLORENCIA CASTELLO; DOMINGO PALMERO; AGUSTÍN ROLANDELLI; JUAN L. IOVANNA; VERÓNICA E. GARCÍA

Mar del Plata

Congreso; LXI Reunión Científica Anual de la Sociedada Argentina de Investigación Clínica (SAIC); 2016

SAIC SAI SAFE

Current IFNγ release assays (IGRAs) cannot discriminate active and latent infection with Mycobacterium tuberculosis (Mtb). Moreover, no gold standard to diagnosis latent tuberculosis (LTBI) is available. Previously, we demonstrated that the dormancy antigen Rv2626c and six peptide-pools derived from its sequence discriminated by IFNγ secretion LTBI subjects from healthy donors (HD) and tuberculosis (TB) patients. Stimulation of peripheral blood mononuclear cells (PBMCs) or whole blood with peptides or Rv2626c induced differential amounts of IFNγ among the groups. The aim of this work was to advance in the development of a diagnostic kit to detect tuberculosis infection. For this, we focused on two main objectives: i) To develop new immunogenic pools with less than six peptides to diminish costs and ii) To use new pools to evaluate their immunogenicity in whole blood incubated in polystyrene tubes (as used by QuantiFERON-TB© - QFT). IFNγ release was measured by ELISA. To design new pools we first stimulated LTBI PBMCs with all the individual peptides (13-15 mers, overlapped by 11 aa; 36 in total, covering the complete Rv2626c sequence). Peptides 1, 2, 5, 11, 12, 22, 23, 24, 30, 33 and 36 were the more immunogenic peptides. Then, we generated the new pools , BD, IM1, IM2, IM3 and PBMCs were stimulated with them (37º, 5d). We found that treatment with these pools significantly differentiate among the groups. Furthermore, when we stimulated whole blood with these pools (37º, 16h) we observed that pool IM2 clearly differentiated LTBI individuals from HD and TB (0.473 ± 0.020 IU/mL of IFNγ (LTBI), 0.095 ± 0.030 (HD), 0.008 ± 0.008 (TB); p