Comparison of molecular and immunological methods for the rapid diagnosis of smear-negative tuberculosis.

SETTING: The rapid diagnosis of pulmonary tuberculosis (TB) can be challenging if acid-fast bacilli are not detected by sputum smear microscopy. OBJECTIVE: To compare the results of the GeneXpert® MTB/RIF assay on a single sputum or bronchoalveolar lavage (BAL) specimen test with local immunodiagnosis from the site of disease using the T-SPOT®.TB assay on BAL (BAL T-SPOT). DESIGN: The Xpert and BAL T-SPOT tests were compared in 96 patients suspected of having sputum smear-negative pulmonary TB admitted to a referral centre in Germany. RESULTS: BAL T-SPOT identified 10 of 11 patients with pulmonary TB (including 3/4 patients with culture-confirmed TB) with a negative Xpert test. Using Xpert, the sensitivity, specificity and positive and negative likelihood ratios (LRs) were respectively 60.0%, 97.4%, 30.0% and 0.4% in culture-confirmed cases and 42.1%, 97.4%, 21.1% and 0.6% in all TB patients. In contrast, using BAL T-SPOT, the sensitivity, specificity and positive and negative LRs were respectively 80.0%, 62.6%, 2.1% and 0.3% in culture-confirmed cases and 89.4%, 62.6%, 2.4% and 0.2% in all TB patients. CONCLUSION: In sputum smear-negative TB suspects, a positive Xpert result is strongly indicative of culture confirmation; however, a negative result is insufficient to rule out active TB. Where clinical suspicion of pulmonary TB persists despite a negative Xpert result, local immunodiagnosis using T-SPOT on BAL may increase diagnostic accuracy.

Impact of a Mycobacterium tuberculosis-specific interferon-γ release assay in bronchoalveolar lavage fluid for a rapid diagnosis of tuberculosis.

OBJECTIVES: Evaluation of different methods for an initial treatment decision in individuals with suspected pulmonary tuberculosis. BACKGROUND: Recently, important advances regarding the diagnosis of pulmonary tuberculosis have been introduced, which influence the decision to initiate anti-tuberculosis treatment. METHODS: To evaluate the impact of different methods for the presumed diagnosis of tuberculosis, individuals with suspected tuberculosis were prospectively enrolled following a specific algorithm including initial smear microscopy and Mycobacterium tuberculosis-specific nucleic acid amplification (NAAT) from sputum. In cases of negative initial test results, tuberculin skin testing, bronchoscopy with transbronchial biopsies and interferon-γ release assays (IGRAs) in peripheral blood and bronchoalveolar lavage (BAL) fluid were performed. RESULTS: Amongst 135 individuals with suspected tuberculosis, 42 had tuberculosis, 10 had nontuberculous mycobacteria pulmonary infection/colonization (one had both tuberculosis and nontuberculous mycobacteria pulmonary infection/colonization) and 84 had an alternative final diagnosis. The sensitivity and specificity were 41% and 99% [positive likelihood ratio (LR+) = 40] for sputum microscopy and 31% and 98% (LR+) = 16) for BAL nucleic acid amplification, respectively. In patients with acid-fast bacilli smear-negative tuberculosis (25/42, 59.5%), M. tuberculosis-specific BAL fluid IGRA was 92% sensitive and 87% specific (LR+) = 7) for the diagnosis of tuberculosis. CONCLUSION: None of the microbiological or immunological methods that aim to provide a rapid diagnosis of tuberculosis whilst waiting the confirmation of the M. tuberculosis culture results is on its own accurate enough to diagnose or exclude pulmonary tuberculosis. Negative sputum microscopy and M. tuberculosis-specific NAAT results should prompt bronchoscopy including BAL for M. tuberculosis-specific IGRA in individuals with suspected pulmonary tuberculosis.

Suttons's law: local immunodiagnosis of tuberculosis.

Recently, important advances have been made in the immunodiagnosis of tuberculosis. New T cell interferon-gamma release assays (TIGRA) are more specific and more sensitive than the tuberculin skin test (TST) for the diagnosis of Mycobacterium tuberculosis (MTB) infection. However, like the TST, TIGRA are unable to distinguish between active tuberculosis (TB), latent TB infection (LTBI) and treated TB if performed on blood mononuclear cells alone. In active TB, MTB-specific T cells are actively recruited to the site of infection and can rapidly be identified in extrasanguinous fluids, such as pleural effusions, ascites, cerebrospinal fluid, and in bronchoalveolar lavages. This review summarizes recent findings comparing systemic and local immune responses against MTB. Although bacteriological and histological methods have the highest specificity for TB in terms of diagnosing active TB and the number of TB patients in whom extrapulmonary TIGRAs have been evaluated is still limited, a comparison of local and systemic MTB-specific immune responses is a promising technique to rapidly distinguish active TB from latent MTB infection in routine clinical practice.

Rapid diagnosis of pulmonary TB by BAL enzyme-linked immunospot assay in an immunocompromised host.

Immunocompromised patients with acid-fast bacilli (AFB) smear-negative active pulmonary tuberculosis (pTB) often present with nonspecific clinical symptoms and findings. T-cell interferon-gamma release assays (TIGRA) performed on whole blood (using ELISA) or peripheral blood mononuclear cells (using enzyme-linked immunospot assay (ELISPOT)) are more sensitive for the diagnosis of Mycobacterium tuberculosis (MTB) infection than the tuberculin skin test (TST), but cannot distinguish active from latent MTB infection. The present authors report a 38-yr-old female presenting with a 3-week history of malaise, dyspnoea, fevers and coughing, who had received immunosuppressive therapies over 8 months for mixed connective tissue disease. Chest radiograph and thoracic computed tomography showed ground glass opacities in both lower lobes. The TST-induration was 0 mm and AFBs or MTB nucleic acid was not detected on sputum and bronchial secretions. However, TIGRAs performed on peripheral blood cells were reactive. A high frequency of MTB-specific T-cells compatible with the immunodiagnosis of active pTB was detected among bronchoalveolar lavage cells using ELISPOT. Antituberculous therapy was initiated 18 days before MTB was discovered on sputum cultures. Detection of Mycobacterium tuberculosis-specific T-cells in the bronchoalveolar lavage using enzyme-linked immunospot assay is a promising tool for the diagnosis of active pulmonary tuberculosis in immunocompromised patients with negative acid-fast bacilli smears.

Local immunodiagnosis of pulmonary tuberculosis by enzyme-linked immunospot.

Lymphocytes are crucial in the immune defence against Mycobacterium tuberculosis (MTB) infection. The aim of the present study was to ascertain whether or not MTB-specific lymphocytes are selectively compartmentalised in the lungs of patients with minimal active pulmonary tuberculosis (PTB). Patients with smear-negative MTB-culture-confirmed PTB were prospectively recruited. Differential cell counts, immunophenotyping with monoclonal antibodies directed against the cell surface markers CD4, CD8, CD4CD45RA, CD4CD45R0, CD38, human leukocyte antigen DR, CD19, CD3, CD57 and CD16 and MTB-specific enzyme-linked immunospot assays of peripheral blood mononuclear cells and bronchoalveolar lavage (BAL) mononuclear cells with 6-kDa early secretory antigenic target and culture filtrate protein 10 were performed. Among 12 patients with culture-confirmed smear-negative PTB, no differences were found in the distribution of total CD4 or CD8 T-cells in peripheral blood or BAL fluid (BALF). Activated human leukocyte antigen-DR-positive cells, as well as memory CD4CD45R0-positive T-cells, were expanded among cells of the BALF. Compared with a group of control patients with alternative pulmonary pathologies, there was no significant difference in lymphocyte subpopulations. However, 6-kDa early secretory antigenic target- and culture filtrate protein 10-specific lymphocytes were more concentrated, with a median BALF:peripheral blood ratio of 9.9 and 8.9, respectively, in patients with PTB. Mycobacterium tuberculosis-specific T-cells are highly selectively compartmentalised at the site of infection in active pulmonary tuberculosis.

Use of a T-cell interferon-gamma release assay for the diagnosis of tuberculous pleurisy.

The diagnosis of pleural tuberculosis (plTB) by the analysis of pleural effusions (PEs) with standard diagnostic tools is difficult. In routine clinical practice, the present authors evaluated the performance of a commercially available Mycobacterium tuberculosis (MTB)-specific enzyme-linked immunospot assay on peripheral blood mononuclear cells (PBMCs) and pleural effusion mononuclear cells (PEMCs) in patients with suspect plTB. The T-SPOT.TB test (Oxford Immunotec Ltd, Abingdon, UK) was performed on PBMCs and PEMCs in 20 patients with a clinical and radiological suspect of plTB and in 21 control subjects with a diagnosis of PE of nontuberculous origin at four centres participating in the European Tuberculosis Network. In total, 18 (90%) out of 20 patients with plTB tested T-SPOT.TB-positive on PBMCs and 19 (95%) out of 20 on PEMCs. Among controls, T-SPOT.TB was positive in seven out of 21 (33%) patients when performed on PBMCs (these patients were assumed to be latently infected with MTB) and five (23%) out of 21 when performed on PEMCs. Sensitivity and specificity of T-SPOT.TB for the diagnosis of active plTB when performed on PEMCs were 95 and 76%, respectively. Enumerating Mycobacterium tuberculosis-specific T-cells in pleural effusion mononuclear cells by ELISPOT is feasible in routine clinical practice and may be useful for a rapid and accurate diagnosis of pleural tuberculosis.