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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

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
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

Tuberculosis (TB) is one of the leading causes of morbidity and mortality worldwide, affecting more than 8 million persons annually (1), mostly with pulmonary tuberculosis (pTB). Confirmation of the presumptive diagnosis of tuberculosis is usually delayed, as detection of growth of Mycobacterium tuberculosis (MTB) in liquid or solid medium takes an average of 2 wk of culture (2). However, only 72% of cases of pulmonary tuberculosis in Germany were culture positive in 2003 (3). The rapid diagnosis of pulmonary tuberculosis relies therefore on clinical symptoms and radiologic findings, and on the detection of acid/alcohol-fast bacilli (AFB) in sputum or bronchial secretions. Detection of MTB-specific gene sequences in respiratory specimens by DNA amplification may rapidly confirm the diagnosis of pTB, but DNA amplification for the diagnosis of tuberculosis is not standardized and is available only in specialized institutions.

The sensitivity of sputum or bronchoalveolar lavage (BAL) microscopy for the detection of AFB is variable in pTB and ranges from 50 to 80% for three consecutive specimens (4). In patients with smear-negative pTB the sensitivity of sputum or BAL MTB-specific DNA amplification ranges from 48 to 80% (5). Thus, a substantial proportion of patients with pTB remain undiagnosed by current laboratory techniques until results of MTB cultures become available.

Enzyme-linked immunospot (ELISPOT) and enzyme-linked immunoassay (ELISA) techniques have been developed to rapidly detect IFN- production by MTB-specific peripheral blood mononuclear cells (PBMCs) for the diagnosis of MTB infection. These assays use peptides from early antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10), which are encoded by the region of difference-1 (RD-1) in the MTB genome, a region that is absent from all strains of Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine and most nontuberculous mycobacteria (6–9). The sensitivity of these tests in active TB ranges from 76 to 100% (9–17) whereas the specificity exceeds 88% (9, 14–16, 18). In TB contact tracing, these tests are a better indicator of close exposure to an index case than the tuberculin skin test (TST) (19–23). Although these tests are unaffected by prior BCG vaccination, they cannot distinguish between active and latent TB when performed on PBMCs (reviewed by Dheda and coworkers [24] and Pai and coworkers [25]).

Approximately 95 to 98% of T lymphocytes are confined to lymphatic organs (26). In tuberculosis, MTB-specific T cells clonally expand and are recruited to the site of infection (27, 28). In persons with pulmonary tuberculosis MTB-specific T cells should therefore be detectable among mononuclear cells derived by BAL of the affected pulmonary segment. In patients with suspected tuberculosis and smear-negative microscopy, bronchoscopy with BAL is a safe procedure when guidelines are followed and may be indicated to exclude other diseases such as bronchogenic carcinoma or sarcoidosis (29, 30). We therefore recruited consecutively all patients with a suspected diagnosis of pTB and smear-negative sputum microscopy and studied prospectively whether patients with smear-negative pTB can be detected rapidly by enumeration of ESAT-6– and CFP-10–specific IFN-–producing T cells from BAL fluid.

To our knowledge, this is the first prospective trial in routine practice to evaluate whether active pTB can be distinguished from latent TB infection (LTBI) by enumeration of MTB-specific T cells from the site of disease.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Patients
After informed consent was obtained, patients with three negative AFB smear results on consecutive days, pulmonary infiltrates by chest X-ray or thoracic computed tomography (CT), and a medical history compatible with TB were enrolled at the Medical Clinic of the Research Center Borstel (Borstel, Germany) and the Hospital Großhansdorf (Großhansdorf, Germany). PBMCs were obtained by a venous blood draw of 30 ml for two-color flow cytometry and MTB-specific ELISPOT. Bronchoscopy was performed according to German guidelines (31) with transbronchial biopsies for histology and BAL with 200–300 ml of normal saline from an affected lung segment for microbiological culture, MTB-specific DNA amplification (BD ProbeTec ET system; BD Diagnostic Systems, Sparks, MD), cytological differentiation, and BAL ELISPOT.

Tuberculin skin testing was performed with 0.1 ml (2 TU) of tuberculin RT23 (Statens Serum Institut, Copenhagen, Denmark). The induration was measured after 48–72 h, using the ballpoint technique (32).

Test results for three initial sputum samples and sputum or BAL MTB-specific DNA amplification were obtained for 100 consecutive patients with culture-confirmed pTB at the Medical Clinic of the Research Center Borstel.

The study was approved by the ethics committee of the University of Luebeck Medical School (Luebeck, Germany), and all patients gave written, informed consent.

Immunophenotyping
Lymphocyte subsets were enumerated in PBMCs obtained from freshly drawn blood and in cells from BAL fluid, using directly labeled murine monoclonal antibodies against CD3, CD4, CD8, CD45R0, CD45RA, HLA-DR, and CD38 (Dako, Hamburg, Germany) by two-color flow cytometry (FACSCalibur; Becton Dickinson, Heidelberg, Germany).

Cell Cultures
PBMCs were prepared by Ficoll-Hypaque density gradient centrifugation from heparinized blood. Single-cell suspensions from BAL fluid were obtained by passing the BAL fluid through a stainless steel sieve (WMF, Teesieb Profi Plus; WMF, Geislingen, Germany) with a mesh aperture of 0.5 mm.

Cells were put into culture in RPMI 1640 medium containing penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mmol/L), and 5% fetal calf serum (culture medium).

ELISPOT Assay
ELISPOT assays for human IFN- were performed with test plates from the T-SPOT.TB test (Oxford Immunotec, Abingdon, UK). Briefly, 200,000 PBMCs and mononuclear cells from BAL fluid (BALMCs) were plated overnight on 96-well plates, which had been precoated with a mouse anti-human IFN- antibody, in 200 µl of culture medium per well. The cells were left unstimulated (negative control) or were stimulated with anti-CD3 monoclonal antibody (clone X35, 10 ng/ml; Beckman Coulter, Krefeld, Germany; positive control), with purified protein derivative of tuberculin (PPD, 10 µg/ml; Statens Serum Institut), or with ESAT-6 and CFP-10 peptides (5 µg/ml; kindly provided by Oxford Immunotec). Culture of the plates, washing, counterstaining, visualization, and analysis of the spots were performed according to manufacturer guidelines.

The response of stimulated cultures was considered positive when the test well contained at least five more spots and had twice the number of spots than the control well. The background number of spots in negative control wells was always less than five spots per well.

Statistical Analysis
All statistical tests were performed as exploratory analyses without adjustment for multiple testing, with nominal significance defined as p < 0.05. Continuous variables were compared by nonparametric testing (Mann-Whitney test) because the data were not normally distributed.

	RESULTS

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After informed consent was obtained, 37 patients with a suspected diagnosis of pTB were prospectively enrolled in this study. In eight of the patients, MTB was cultured from either sputum, BAL fluid, or both. In addition, four patients who presented with pulmonary infiltrates typical of TB on chest CT responded clinically and radiographically to anti-TB therapy and were given a final diagnosis of pTB (Table 1).

Patients with TB were younger (31.1 vs. 50.5 yr) when compared with patients with alternative diseases (Table 2). The median induration of the TST was 20 mm in the TB group and 0 mm in the non-TB group (Figure 1). Two patients (17%) in the TB group had a TST induration of 5 mm or less and 3 of 25 patients (12%) in the control group had a TST induration of 15 mm or more. The TST was not repeated in two patients with a previous history of TB. In the TB group. MTB-specific DNA amplification was positive only in the sputum of two patients, in the sputum and BAL fluid of one patient, and only in the BAL fluid of one patient. In the control group, two patients with pulmonary infiltrates due to bronchogenic carcinoma also had a positive MTB-specific DNA amplification result from BAL fluid. Both patients had a history of pTB in 1994 and 2005, respectively, and each was treated for 6 mo at that time.

View larger version (6K): [in this window] [in a new window]   Figure 1. Induration diameters of tuberculin skin tests (TSTs) of patients with smear-negative pulmonary tuberculosis (TB; n = 12, open circles) or patients with pulmonary infiltrates of other origin (n = 25, solid triangles). Seven patients in the non-TB group had been treated for pulmonary TB in the past.

In 100 patients (29 females and 71 males) with culture-confirmed pTB identified retrospectively at our institution, AFB were seen on sputum microscopy in 44, 51, and 54 instances with the first, second, and third examined sputum sample, respectively. In 49 patients, MTB-specific DNA amplification was performed on sputum, bronchial secretion, or gastric aspirate. Positive results were found in 41 patients (84%) and in 18 of 25 patients (72%) with smear-negative TB. Of those 18 smear-negative patients with positive DNA amplification results, 11 were positive on sputum, 4 were positive on BAL, one was positive on gastric aspirate, and 2 had a positive result from a transbronchial biopsy specimen.

Among the 37 prospectively recruited patients, all 12 patients with TB but none of the control subjects had more than five spot-forming cells per 200,000 BALMCs with either antigen in the BAL ELISPOT. Median ESAT-6– and CFP-10–specific ELISPOT responses (CFP-10–specific responses could not be performed in 3 of 25 patients from the control group because of a technical error) were higher in PBMCs (17.0 and 24.5) and BALMCs (37.5 and 49.5) from patients with TB versus control subjects (PBMCs, 1.0 and 1.0; BALMCs, 0 and 0; respectively; Table 1 and Figure 2). The sensitivity and specificity of the MTB-specific ELISPOT with ESAT-6 and CFP-10 peptides performed on BALMCs were both 100% for the diagnosis of active tuberculosis (95% confidence interval, 0.97–1.0).

View larger version (12K): [in this window] [in a new window]   Figure 2. Concentration of early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) IFN-–producing spot-forming cells (sfc) in peripheral blood mononuclear cells (PBMCs) or bronchoalveolar lavage mononuclear cells (BALMCs) of patients with smear-negative pulmonary TB (n = 12, open and solid circles for ESAT-6 and CFP-10 peptides, respectively) or patients with nontuberculous pulmonary infiltrates (n = 25; open and solid diamonds for ESAT-6 and CFP-10 peptides, respectively). The dashed line represents a cutoff of 5 sfc/200,000 cells. In contrast to ELISPOT performed on PBMCs, ELISPOT performed on BALMCs fully discriminates between patients with active pulmonary TB and patients with nontuberculous pulmonary infiltrates. All spots are documented after subtraction of spots in negative control. Bars represent median values.

When instead of ESAT-6 and CFP-10 peptides, PPD was used as an antigen, results were similar but did not fully discriminate between patients with active pTB and control patients. In the control group, BAL specimens from seven patients were positive for PPD (Figure 3).

View larger version (9K): [in this window] [in a new window]   Figure 3. Concentration of purified protein derivate (PPD)–specific IFN-–producing spot-forming cells in PBMCs or BALMCs of patients with smear-negative pulmonary TB (n = 12; open triangles) or patients with nontuberculous pulmonary infiltrates (n = 25; solid triangles). The dashed line represents a cutoff of 5 sfc/200,000 cells. In contrast to ELISPOT performed on PBMCs and BALMCs with ESAT-6 and CFP-10 (see Figure 2), a PPD-based ELISPOT performed on PBMCs or BALMCs does not fully discriminate between patients with active pulmonary TB and patients with nontuberculous pulmonary infiltrates. All spots are documented after subtraction of spots in negative control. Bars represent median values.

Numbers of ESAT-6–, CFP-10–, and PPD-specific spot-forming cells among BALMCs, but not among PBMCs, were significantly associated with pTB independent of age and sex (data not shown).

There were no differences in the distribution of CD4⁺, CD8⁺, CD38⁺, HLA-DR⁺, CD4⁺CD45RO⁺, or CD4⁺CD45RA⁺ T cells from the blood or BAL among patients from both groups (data not shown).

	DISCUSSION

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MTB-specific IFN- release assays based on MTB RD1 peptides ESAT-6 and CFP-10 have been developed for commercial use both as ELISPOT and ELISA for the diagnosis of TB and LTBI from blood. The ELISPOT assay has higher sensitivity and specificity (9, 15, 25, 33–41) for the diagnosis of TB compared with the TST. In contrast to the TST, the MTB-specific IFN- release assays are not influenced by prior BCG vaccination and have lower cross-reactivity to mycobacteria other than TB (15, 21, 42, 43). However, like the TST, MTB-specific IFN- release assays do not allow distinction between active TB and LTBI when performed on blood.

Smear-negative TB is a common problem in clinical practice, where a clear and rapid distinction between active TB and LTBI has not been possible so far. In our hospital, 54% of patients with pTB confirmed by culture could not be identified by three consecutive sputum examinations for the presence of AFB. Moreover, the sensitivity of an MTB-specific polymerase chain reaction in culture-positive smear-negative pTB was only 64% (95% confidence interval, 0.58–0.76) in our study (3 of 8 prospectively studied patients and 18 of 25 retrospectively studied patients), comparable to a sensitivity of 58–86%, which has been published by others (44, 45).

In the diagnosis of MTB infection, the MTB-specific ELISPOT assay (T-SPOT.TB) has a high diagnostic sensitivity and a low rate of indeterminate results when performed on PBMCs (46, 47). For this test, measured IFN- release can be directly related to the number of antigen-specific mononuclear cells in a biological specimen. In TB, antigen-specific T cells clonally expand and are recruited to the sites of infection (27, 28). MTB antigen–specific lymphocytes are compartmentalized in the lungs in healthy household contacts (48) and in patients with active pTB and on challenge produce helper T-cell type 1 cytokine host responses (49). In an experimental setting, helper T-cell type 1–like effector memory cell responses can be elicited in BALMCs after bronchoscopic challenge with PPD into a lung segment of healthy TST-positive volunteers (50, 51). Measurement of type 1 cytokine production by CD4⁺ T cells in response to PPD in BAL fluid was shown to be highly sensitive and specific for the diagnosis of active TB (52). However, the analysis of RD-1–specific immune responses of mononuclear cells from the site of MTB infection may be more specific for the diagnosis of active TB than analysis of PPD responses. For example, Wilkinson and coworkers showed that ESAT-6–specific IFN-–secreting T cells are concentrated in tuberculous pleural effusion and ascites and are absent in pleural effusions of other etiology (53). The clinical utility of this approach was demonstrated in the diagnosis of a patient with disseminated pleural TB and hairy cell leukemia (54).

Although numbers of PPD-reactive BALMCs were much higher in patients with pTB than in control subjects, some patients with nontuberculous pulmonary infiltrates also had high numbers of PPD-specific IFN-–releasing cells in the BAL fluid. The ELISPOT assay of BALMCs with ESAT-6 and CFP-10 peptides was therefore more specific for the diagnosis of pTB than was the ELISPOT with PPD peptide from BAL fluid, the MTB-specific ELISPOT performed on PBMCs, or the TST. We found 10 patients in the control group with positive IFN- responses to ESAT-6 and/or CFP-10 peptides in PBMCs. Five had a history of prior TB and one had received prior treatment for LTBI. The remaining four patients in the control group with positive IFN- responses to ESAT-6 and/or CFP-10 in the blood and not BAL fluid were suspected to be latently infected with MTB.

In this study, we found that both ESAT-6 and CFP-10 peptide–specific mononuclear cell responses were detectable in BALMCs only from patients with active pTB, whereas these responses were absent in patients with LTBI, in patients with a history of TB without current reactivation, or in patients with pulmonary infiltrates of other origin. By enumerating ESAT-6– and CFP-10–specific BALMCs, patients with active TB could be fully distinguished from patients with pulmonary infiltrates of other origin.

Thus, active pTB may be better diagnosed when organ-specific immune responses with MTB-specific peptides are evaluated by ELISPOT.

However, our study has several limitations. The proportion of male patients was higher and patients were younger in the TB group compared with the control group. Therefore, the influence of age or sex for MTB-specific immune responses by BALMCs cannot be excluded.

Although the diagnosis of active pTB could not be verified by a positive MTB culture result from the sputum or the BAL fluid of four patients, the diagnosis of pTB in these cases was likely, given that all patients had positive TSTs and typical pulmonary infiltrates on chest CT suggesting active TB, and all four patients responded well to anti-TB therapy. All four patients also had positive ESAT-6– and CFP-10–specific ELISPOT on PBMCs.

When a cutoff of five antigen-reactive cells per 200,000 BALMCs was used, the sensitivity and specificity of the MTB-specific ELISPOT with ESAT-6 and CFP-10 peptides were both 100% (95% confidence interval, 0.97–1.0) in this small cohort. These values are much higher than the sensitivity and specificity values of sputum or BAL DNA amplification, but these data will need validation in larger studies, including countries with a high TB incidence. It also remains to be seen whether a BAL ELISPOT can be applied to patients with immunodeficiencies such as HIV infection, where lower numbers of BALMCs may be expected. If confirmed in larger cohorts, the findings of this study may potentially improve the diagnostic evaluation of smear-negative pulmonary tuberculosis in clinical settings, where bronchoscopy and ELISPOT techniques are available.

We have shown for the first time that the diagnosis of smear-negative pTB can be established rapidly by enumeration of MTB-specific T cells from BAL fluid and that the MTB-specific ELISPOT, when applied to BAL fluid, may distinguish with high sensitivity and specificity between active pTB and LTBI.

	Acknowledgments

 
The authors thank Dr. Sabine Ruesch-Gerdes and Dr. Elvira Richter (National Reference Center for Mycobacteria, Borstel, Germany) for assistance and critical review of the manuscript, and Silke Lange (Dortmund, Germany) for statistical advice.

	FOOTNOTES

 
Supported by the H.W. and J. Hector Foundation (C.L.). A.L. is a Wellcome Senior Research Fellow in Clinical Sciences.

Originally Published in Press as DOI: 10.1164/rccm.200604-465OC on July 20, 2006

Conflict of Interest Statement: C.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.E. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. U.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.L. is a named inventor on several patents related to T-cell diagnosis filed by the University of Oxford. Regulatory approval of ELISPOT has been undertaken by Oxford Immunotec, in which he is a shareholder and to which he acts as a scientific advisor. C.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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