title: Diagnosis of Mycobacterium tuberculosis infection using ESAT-6 and intracellular cytokine cytometry.
abstract: Diagnosis of infection with Mycobacterium tuberculosis (MTB) using tuberculin skin testing (TST) is often hampered by prior Bacille Calmette-Guérin (BCG) vaccination. ESAT-6 is a protein that is expressed by MTB but absent in BCG. It has been postulated that it might be useful in distinguishing MTB-specific immune responses. This study measured CD4 T cell responder frequencies specific for ESAT-6 and the TST reagent purified protein derivative (PPD) in patients with tuberculosis (n = 16), controls with non-tuberculous pneumonia (n = 8) and normal subjects (n = 7). Responses were identified using the intracellular cytokine staining technique and flow cytometry on whole blood samples, and performed blinded to the patient condition. Antigen-specific CD4 cells were defined by CD69 positivity and one or more cytokine [interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma] and/or CD40L positivity. With ESAT-6 stimulation it was found that TB patients had significantly higher frequencies of IFN-gamma and CD40L-positive CD4 T cells compared to the normal group, while no significant differences were measured with PPD stimulation. A responder frequency of 0.01% or higher for at least one of the measured cytokines/CD40L was defined as a positive response. Using this criterion to compare the two patient groups, PPD had 100% sensitivity but 0% specificity while ESAT-6 had 100% sensitivity and 88% specificity. Use of MTB-specific proteins such as ESAT-6 in combination with intracellular cytokine staining and flow cytometry has the potential to identify individuals with MTB infection.
authors.affiliation: Department of Respiratory Medicine, Monash Medical Centre, Melbourne, Victoria, Australia.
authors.forename: A J
authors.initials: AJ
authors.lastname: Hughes
authors.forename: P
authors.initials: P
authors.lastname: Hutchinson
authors.forename: T
authors.initials: T
authors.lastname: Gooding
authors.forename: N J
authors.initials: NJ
authors.lastname: Freezer
authors.forename: S R
authors.initials: SR
authors.lastname: Holdsworth
authors.forename: P D R
authors.initials: PD
authors.lastname: Johnson
mesh_terms: D000328:Adult; D000368:Aged; D000942:Antigens, Bacterial / Q000276:immunology; D001426:Bacterial Proteins; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D023201:CD40 Ligand / Q000276:immunology; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000379:methods; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D007378:Interleukins / Q000276:immunology; D018655:Lymphocyte Count; D008297:Male; D008875:Middle Aged; D014373:Tuberculin; D014376:Tuberculosis / Q000175:diagnosis* / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D016207:Cytokines; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D000939:Epitopes; D007378:Interleukins; D014373:Tuberculin; D023201:CD40 Ligand; D007371:Interferon-gamma