title: Feasibility and diagnostic utility of antigen-specific interferon-gamma responses for rapid immunodiagnosis of tuberculosis using induced sputum.
abstract: BACKGROUND
  The diagnosis of smear-negative or sputum-scarce tuberculosis (TB) is problematic as culture takes several weeks and representative biological samples are difficult to obtain. RD-1 antigen-specific interferon-gamma release assays (IGRAs) are sensitive and specific blood-based tests for the diagnosis of M. tuberculosis infection. The feasibility and diagnostic utility of this rapid immunodiagnostic assay, using cells from induced sputum, is unknown.
  
  
  RESULTS
  Cells isolated from induced sputum were co-cultured with ESAT-6 and CFP-10 antigens using a standardized enzyme-linked immunospot (ELISPOT) assay (T-SPOT.TB) in 101 consecutively recruited TB suspects or non-TB controls. An optimization phase using 28 samples was followed by a validation phase using samples from 73 participants (20 with definite or probable TB, and 48 with non-TB). Despite optimization of sputum processing 65/73 (89%) of the IGRAs in the validation phase were inconclusive. 44/73 (60%) tests failed due to sputum induction-related factors [sputum induction-related adverse events (n = 5), inadequate sputum volume (n = 8), non-homogenisable sputum (n = 7), and insufficient numbers of cells to perform the assay (n = 24)], whilst 20/73 (27%) tests failed due T-SPOT.TB assay-related factors [excessive debris precluding reading of spots in the ELISPOT well (n = 6), failure of the positive control (n = 11), or high spot count in the negative control (n = 3)]. Only 8/73 (11%) of the available samples could therefore be correctly categorized (7 definite or probable TB, and 1 non-TB patient). Thus, 13/20 (65%) of the definite or probable TB cases remained undiagnosed.
  
  
  CONCLUSIONS
  Rapid immunodiagnosis of pulmonary TB by antigen-specific IFN-gamma ELISPOT responses, using cells from induced sputum, is possible. However, the test, in its current ELISPOT format, is not clinically useful because the majority of the assays are inconclusive.
authors.affiliation: Lung Infection and Immunity Unit, Division of Pulmonology, UCT Lung Institute and Clinical Immunology, Department of Medicine, University of Cape Town, Cape Town, South Africa.
authors.forename: Tamaryn J
authors.initials: TJ
authors.lastname: Cashmore
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Peter
authors.forename: Richard N
authors.initials: RN
authors.lastname: van Zyl-Smit
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Semple
authors.forename: Alice
authors.initials: A
authors.lastname: Maredza
authors.forename: Richard
authors.initials: R
authors.lastname: Meldau
authors.forename: Alimuddin
authors.initials: A
authors.lastname: Zumla
authors.forename: Barbara
authors.initials: B
authors.lastname: Nurse
authors.forename: Keertan
authors.initials: K
authors.lastname: Dheda
mesh_terms: D000328:Adult; D000368:Aged; D000941:Antigens; D016022:Case-Control Studies; D018920:Coculture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D005240:Feasibility Studies; D005260:Female; D006801:Humans; D007159:Immunologic Tests / Q000379:methods*; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology; D008297:Male; D008875:Middle Aged; D013183:Sputum; D014376:Tuberculosis / Q000175:diagnosis*; D055815:Young Adult
chemical_list: D000941:Antigens; D007371:Interferon-gamma