The role of flow cytometry in the interferon-gamma-based diagnosis of active tuberculosis and its coinfection with HIV-1--A technically oriented review.
TB remains uncontrolled. In resource-rich countries, only approximately 60% of diagnoses are confirmed by culture. The number is lower in resource-poor environments. Huge scope therefore exists for alternative diagnostic strategies. Counting antigen-specific lymphocytes by virtue of cytokine production following 8-16 h stimulation with tuberculosis antigens is currently the strategy of choice. Several methods exist, including ELISA, ELISpots, and flow cytometry. Although it is clear that blood samples stimulated by ESAT-6 and CFP-10 antigens discriminate between TB infection and BCG vaccination, it is flow-cytometry that seems to be able to distinguish active TB disease from mere TB exposure. Of the various flow-protocols including four-color tests (CD45-CD3-CD4-IFNgamma), three-color tests (CD3-CD4-IFNgamma) and two-color tests (CD4-IFNgamma), even the simplest is performing well, provided that the results are expressed as percentage of IFN-gamma+ cells per CD4+ lymphocytes (%IFNgamma/CD4+). Studies using broncho-alveloar lavage (BAL) and Induced-Sputum (ISp) show that TB-specific CD4+IFN-gamma+ T cells accumulate in the lung in pulmonary and extra-pulmonary TB at frequencies >5-20-fold more frequent than in blood. This pulmonary homing is absent following BCG immunization. The use of PPD to stimulate CD4+IFN-gamma+ cells in the lung in active TB leads to >3-12-fold greater responses than seen with CFP-10 or ESAT-6, and any interference from BCG vaccination is absent. This method is unaffected by HIV coinfection, which has always been the problem for other immune-based diagnostics. Further, lung-based samples provide material for rapid tests of both the IFN-gamma assay and bacteriology, and importantly, these tests are amenable for future simplification with automated fluorescence-image cytometers.Another development of the multiparameter analytical power of flow-cytometry is to use markers for "lung-seeking" populations of CD4+ T cells in blood, obviating lung sampling. In active TB, but not in BCG vaccinees, TB-specific memory CD4+ T cells can be found in blood that are dominantly CD27-negative and probably lung seeking and can be diagnostically useful.
Janossy
George
G
Department of Immunology, Royal Free and University College Medical School, London, United Kingdom. janossy@rfhsm.u-net.com
Barry
Simon M
SM
Breen
Ronan A M
RA
Hardy
Gareth A D
GA
Lipman
Marc
M
Kern
Florian
F
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D005434:Flow Cytometry / Q000379:methods*; D015658:HIV Infections / Q000150:complications* / Q000175:diagnosis; D006801:Humans; D007371:Interferon-gamma; D014373:Tuberculin / Q000097:blood; D014397:Tuberculosis, Pulmonary / Q000150:complications* / Q000175:diagnosis* / Q000473:pathology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D014373:Tuberculin; D007371:Interferon-gamma