Lymphocyte activation test for diagnosis of seronegative brucellosis in humans.

Seronegative brucellosis is occasionally encountered in clinical practice especially in localized disease where diagnosis is reached mainly through positive blood culture. Cellular immune responses are pivotal for protection against intracellular bacteria such as Brucella. This study was performed to evaluate the expression of activation markers on peripheral blood mononuclear cells in response to in vitro stimulation by whole-cell suspension of Brucella melitensis for the diagnosis of brucellosis. Fifteen seronegative patients with positive blood cultures for Brucella and twenty-five unexposed healthy blood donors serving as controls were recruited for the study. Peripheral blood mononuclear (PBMC) cells were obtained by the method of Ficoll Hypaque density gradient and stimulated in vitro with Brucella antigen. Expression of activation markers was assessed by flow cytometry after staining of PBMC with mononuclear cells with relevant monoclonal antibodies. Incubation with mitogen induced expression of all the four markers was demonstrated in all blood samples. In contrast, samples from all patients of Brucellosis showed significant positive responses with the expression of activation markers (CD38, CD69, CD25, and CD71) on both CD4+ and CD8+ cells as compared with the control group (P < 0.001). It is inferred that there was a remarkable upregulation of activation markers on CD4+ and CD8+ in seronegative patients with Brucellosis. It is recommended that the method can be utilized as a novel diagnostic test for detection of brucellosis where serology is negative.