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Rapid Diagnosis of Smear-negative Tuberculosis by Bronchoalveolar Lavage Enzyme-linked Immunospot

Division of Clinical Infectious Diseases and Division of Immune Cell Analytics, Research Center Borstel, Borstel; Department of Public Health, University of Düsseldorf, Düsseldorf; Center for Pulmonary Medicine and Thoracic Surgery, Hospital Großhansdorf, Großhansdorf, Germany; and Tuberculosis Immunology Group, Nuffield Department of Clinical Medicine, John Radcliff Hospital, University of Oxford, Oxford, United Kingdom

Correspondence and requests for reprints should be addressed to Christoph Lange, M.D., Ph.D., Division of Clinical Infectious Diseases, Research Center Borstel, Parkallee 35, 23845 Borstel, Germany. E-mail: clange{at}fz-borstel.de

Rationale: In a large proportion of patients with active pulmonary tuberculosis (pTB), acid-fast bacilli smear results for sputum and bronchial secretions are negative. Detectable growth of Mycobacterium tuberculosis (MTB) in cultures takes several weeks and MTB-specific DNA amplification results on sputum and bronchial secretions are variable in these patients.

Objective: We investigated whether a rapid diagnosis of pTB can be established by enumeration of MTB-specific mononuclear cells from bronchoalveolar lavage (BAL) fluid in routine clinical practice.

Methods: Patients presenting to a tertiary hospital with medical histories and pulmonary infiltrates compatible with tuberculosis, and negative acid-fast bacilli smear results (three) from sputum, were prospectively enrolled in this study. An MTB-specific enzyme-linked immunospot assay (ELISPOT [T-SPOT.TB; Oxford Immunotec, Abingdon, UK]) with early antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) peptides was performed on peripheral blood mononuclear cells (PBMCs) and mononuclear cells from the BAL fluid (BALMCs).

Measurements and Main Results: Of 37 patients, 12 were found to have smear-negative pTB and 25 were found to have an alternative diagnosis. Patients with tuberculosis had a median number of 17 ESAT-6–specific cells and 24.5 CFP-10–specific cells per 200,000 PBMCs and 37.5 ESAT-6–specific cells and 49.5 CFP-10–specific cells per 200,000 cells in the BAL fluid. Control patients had a median of 1 ESAT-6–specific cell and 1 CFP-10–specific cell per 200,000 PBMCs and no ESAT-6– and CFP-10–specific cells per 200,000 cells in the BAL fluid (p < 0.0001). All patients with TB but none of the control subjects had more than 5 spot-forming cells per 200,000 BALMCs with either peptide in the BAL fluid ELISPOT.

Conclusion: Smear-negative pulmonary tuberculosis can be diagnosed rapidly by identification of MTB-specific cells in the BAL fluid.

Key Words: bronchoalveolar lavage • CFP-10 • ELISPOT • ESAT-6 • tuberculosis

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