Tuberculosis has traditionally been confirmed by AFB staining or culturing Mycobacterium tuberculosis from clinical specimens. However, because of the low sensitivity of the sputum smear examination and of the delayed report in culturing, the conventional methods for detection of M. tuberculosis have not been always satisfactory. In an attempt to develop an alternate tool, this study was initiated to produce monoclonal antibodies (MAb) to lipoarabinomannan B (LAM-B) antigen and to use the antibodies in detecting mycobacteria. In this study, five monoclonal antibodies specific to LAM-B were produced; LAM701 (IgG3), LAM138 (IgM), LAM204 (IgM), LAM302 (IgM), and LAM604 (IgM). A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting LAM-B and other mycobacterial antigens using the monoclonal antibodies. With the MAb LAM701, the minimal detectable concentration was 1.0 ng/ml for LAM-B, and 1.0 microgram/ml for M. tuberculosis whole cells, respectively. When 14 clinical specimens proven to contain AFB by smear staining or culture were examined, ten (71.4%) were positive by the sandwich ELISA; in contrast, sputum smear examination gave positive results in only six (42.9%) specimens. Meanwhile, none of 25 specimens with no evidence of AFB were positive by the sandwich ELISA using the MAb LAM701. Although further evaluations are required, this study suggests that the monoclonal antibodies to LAM-B may be useful in detecting mycobacteria from clinical specimens.
Antibodies, Monoclonal/*biosynthesis ; Antigens, Bacterial/*analysis ; Enzyme-Linked Immunosorbent Assay ; Human ; Lipopolysaccharides/chemistry/*immunology ; Mycobacterium tuberculosis/*immunology ; Sputum/*microbiology ; Support, Non-U.S. Gov't ; Tuberculosis/*diagnosis/microbiology

aboratory diagnosis of tuberculosis (TB) traditionally relies on smear microscopy and culture of Mycobacterium tuberculosis from clinical samples. With recent advances in technology, there have been numerous efforts to develop new diagnostic tests for TB that overcome the low sensitivity and specificity and long turnover time associated with current diagnostic tests. Molecular biological tests based on nucleic acid amplification have brought an unprecedented opportunity for the rapid and specific detection of M. tuberculosis from clinical specimens. With automated sequencing analysis, species identification of mycobacteria is now easier and more accurate than with conventional methods, and rapid detection of mutations in the genes associated with resistance to TB drugs provides early information on the potential drug resistance for each clinical isolate or for clinical samples. In addition, immunological tests for the detection of M. tuberculosis antigens and antibodies to the antigens have been explored to identify individuals at risk of developing TB or with latent TB infection (LTBI). The recent introduction of commercial IFN-gamma assay kits for the detection of LTBI provides a new approach for TB control even in areas with a high incidence of TB. However, these molecular and immunological tools still require further evaluation using large scale cohort studies before implementation in TB control programs.
Antigens, Bacterial/immunology ; DNA, Bacterial/chemistry/genetics ; Humans ; Immunologic Tests/*methods ; Interferon-gamma/analysis ; Mycobacterium tuberculosis/genetics/immunology ; Sequence Analysis, DNA ; Tuberculin Test ; Tuberculosis/*diagnosis/immunology/microbiology

aboratory diagnosis of tuberculosis (TB) traditionally relies on smear microscopy and culture of Mycobacterium tuberculosis from clinical samples. With recent advances in technology, there have been numerous efforts to develop new diagnostic tests for TB that overcome the low sensitivity and specificity and long turnover time associated with current diagnostic tests. Molecular biological tests based on nucleic acid amplification have brought an unprecedented opportunity for the rapid and specific detection of M. tuberculosis from clinical specimens. With automated sequencing analysis, species identification of mycobacteria is now easier and more accurate than with conventional methods, and rapid detection of mutations in the genes associated with resistance to TB drugs provides early information on the potential drug resistance for each clinical isolate or for clinical samples. In addition, immunological tests for the detection of M. tuberculosis antigens and antibodies to the antigens have been explored to identify individuals at risk of developing TB or with latent TB infection (LTBI). The recent introduction of commercial IFN-gamma assay kits for the detection of LTBI provides a new approach for TB control even in areas with a high incidence of TB. However, these molecular and immunological tools still require further evaluation using large scale cohort studies before implementation in TB control programs.
Antigens, Bacterial/immunology ; DNA, Bacterial/chemistry/genetics ; Humans ; Immunologic Tests/*methods ; Interferon-gamma/analysis ; Mycobacterium tuberculosis/genetics/immunology ; Sequence Analysis, DNA ; Tuberculin Test ; Tuberculosis/*diagnosis/immunology/microbiology

OBJECTIVETo screen the specific antibody binding peptides of tuberculosis from phage-displayed random phage display (Ph.D.)-7 peptide libraries with purified IgG from tuberculosis serum, and to provide the basis for the development of serological detection reagent of tuberculosis.

METHODSPurified IgG of tuberculosis serum was used as solid ligand to screen the binding peptide from the Ph.D.-7 peptide library, according to the biopanning process of absorption, elution and amplification. Purified IgG of health people serum was used as the molecule of counter selection during the second and third selection. Phages were enriched after 3 rounds of screening, then 20 single phages separately eluted by IgG of tuberculosis and health people were randomly selected on each direction of the determination plates and amplified. The single chains DNA were extracted as template for sequencing. The combination abilities of selected clones to IgG of tuberculosis and health people were tested by indirect enzyme linked immunosorbent assay (ELISA), and the positive clone was identified. Serum samples, from 47 patients with tuberculosis and 37 healthy people vaccinated with BCG, were verified by positive phage clones using phage-ELISA. The verifying results of 24 serum samples used for panning were separately analyzed statistically.

RESULTSAfter 3 rounds of panning, remarkable enrichment of phages that could specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 12 sequences were obtained. 12 phage clones with different sequences were amplified and detected with indirect ELISA and single phage H12 showed higher affinity with IgG of tuberculosis (S/N ≥ 2.1) and was identified as the positive clone. It was found that, in indirect ELISA with single Phage H12, the A(450) value of tuberculosis patients (0.105 ± 0.010) was significantly higher than that of healthy individuals (0.070 ± 0.005), and the t value was 2.912 (P < 0.0001). The A(450) value of 12 serum samples of tuberculosis patients and 12 samples of health individuals used for panning were 0.144 ± 0.016 and 0.052 ± 0.004, and the t value was 5.69 (P < 0.0001).

CONCLUSIONBy using phage-displayed random peptide libraries, we obtained the specific antibody binding peptides of tuberculosis, which showed specific binding activity with IgG of tuberculosis. It can provide a basis for the establishment of a new serological detection method of tuberculosis with peptide.

Adult ; Case-Control Studies ; Female ; Humans ; Immunoglobulin G ; blood ; Male ; Middle Aged ; Molecular Sequence Data ; Mycobacterium tuberculosis ; immunology ; Peptide Library ; Peptides ; immunology ; Tuberculosis ; diagnosis ; immunology ; microbiology ; Young Adult

BACKGROUNDT-SPOT.TB is a novel test for tuberculosis infection with higher sensitivity and specificity than the traditional tuberculin skin test (TST). However, there are no longitudinal data in the literature evaluating T-SPOT.TB for Mycobacterium tuberculosis in patients with acquired immune deficiency syndrome (AIDS) on highly active antiretroviral therapy (HAART). The objective of this study was to assess the value of T-SPOT.TB longitudinally in AIDS patients on HAART without prophylaxis for tuberculosis.

METHODSA prospective observational study was conducted in 50 AIDS patients on HAART. None of the subjects had evidence of active tuberculosis. T-SPOT.TB, a T-cell-based interferon gamma released assay, was performed at the onset of the study and repeated 24 months thereafter. Subjects were evaluated every 6 months during the 36-month follow-up.

RESULTSTwenty-one (42%) AIDS patients on HAART tested positive by T-SPOT.TB (95%CI 28.3% - 55.7%). The pooled spot-forming cells of early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) peptides were 68/million peripheral blood mononuclear cell (PBMC) (interquartile range 44 - 220). The average number of CD4 cells in subjects was (305 +/- 152) cells/microl, and there was no significant difference in T-SPOT.TB response rates between subjects with CD4 cell counts < 200 cells/microl (7/15 (46.7%), 95%CI 21.5% - 71.9%) and those with CD4 cell counts >/= 200 cells/microl (14/35 (40.0%), 95%CI 23.8% - 56.2%, P = 0.662). In the 32 subjects who completed the 24-month follow-up, 10 underwent T-SPOT.TB reversion, one had T-SPOT.TB conversion, six remained positive and 15 remained negative. None of them advanced to active tuberculosis during the 36-month follow-up.

CONCLUSIONThe inactive status of tuberculosis infection may be maintained for a long period in AIDS patients on HAART.

Acquired Immunodeficiency Syndrome ; drug therapy ; immunology ; microbiology ; Adult ; Antiretroviral Therapy, Highly Active ; Female ; Humans ; Interferon-gamma ; secretion ; Male ; Middle Aged ; Mycobacterium tuberculosis ; pathogenicity ; Prospective Studies ; Tuberculosis ; diagnosis ; immunology

Recent increase in the incidence of lung cancer often makes it difficult to differentiate between lung cancer and tuberculosis (TB), due to their radiologic similarities. Fine needle aspiration biopsy (FNAB) has been widely employed for the diagnosis of lung cancer and TB, but the diagnostic accuracy of TB is not high enough. As a rapid screening test for tuberculosis, we evaluated serological tests using Mycobacterium tuberculosis PPD and lipoarabinomannan (LAM) antigens. A total of 95 patients with indication of FNAB cytology from initial CT findings were enrolled. 25 patients had TB, 76 thoracic malignancy, and six (7.9%) of the lung cancer patients also had TB, indicating much higher prevalence of TB in thoracic tumor patients. Antibodies to PPD were elevated in 18 (72.0%) of 25 TB patients and in 22 (31.4%) of 70 patients with thoracic malignancy. In contrast, only 3 (4.7%) of 64 healthy controls aged 40 or above were seropositive to PPD antigen. The prevalence of anti-PPD antibodies in thoracic tumor patients was therefore significantly greater than that amongst the healthy controls (p 0.001, chi-square test). However, no significant difference in the prevalence of anti-LAM antibodies was found between study subjects and controls. This study demonstrates that thoracic tumor patients have significantly elevated antibodies to PPD; therefore, high anti-PPD seroreactivity in thoracic tumor patients should be cautiously interpreted. A longitudinal investigation on seropositive thoracic tumor patients is required to determine the role of the serological test for TB in lung cancer patients.
Adult ; Aged ; Aged, 80 and over ; Antibodies, Bacterial/*analysis ; Biopsy, Needle ; Female ; Human ; Lipopolysaccharides/*immunology ; Lung Neoplasms/complications/diagnosis/*microbiology ; Male ; Middle Age ; Mycobacterium tuberculosis/*immunology ; Seroepidemiologic Studies ; Tuberculin/*immunology ; Tuberculosis, Pulmonary/complications/diagnosis

OBJECTIVETo establish a recombinant plasmid of CFP32 of Mycobacterium tuberculosis in E. coli, and to analyze its antigenicity.

METHODSRv0577 gene was amplified by polymerase chain reaction from genome of Mycobacterium tuberculosis, and then cloned into vector pMD18-T followed by the subclone into the expression vector pET21a. Recombinant CFP32 was expressed and purified. The antigenicity of the recombinant protein was analyzed by using Western-blot. The purified recombinant CFP32 protein was used as an antigen to screen the sera of 7 pulmonary TB patients (n = 97), as well as the other pulmonary disease patients (n = 25), and the clinically healthy controls (n = 38) by ELISA.

RESULTSRecombinant plasmid of CFP32 was established, and be expressed efficiently in E. coli BL21 (DE3). The relative molecular mass of the protein was about 300,000 by SDS-PAGE analysis. The protein purified by Ni-NTA was in a purity over 90%, which was confirmed by Western-blot analysis. ELISA analysis showed its sensitivity and specificity were 63.9% (62/97) and 96.8% (2/63) respectively.

CONCLUSIONThe recombinant expression plasmid pET21a CFP32 has been constructed and CFP32 proteins has been successfully expressed and be purified in E. coli and, ELISA analysis has identified the recombinant CFP32 as a candidate antigen for TB serodiagnosis.

Antigens, Bacterial ; blood ; Bacterial Proteins ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; Gene Expression ; Humans ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Plasmids ; Recombinant Proteins ; Serologic Tests ; Tuberculosis, Pulmonary ; diagnosis ; microbiology

The assessment of the adenosine deaminase activity (ADA) in the pleural effusion is used for the diagnosis of tuberculous pleural effusion (TPE). To examine whether the procedure can be applied to immunocompromised patients, we analyzed the ADA activity in the pleural fluid of renal transplant recipients. We studied 23 renal transplant patients with TPE (21 men and 2 women; the mean age, 33 years). They were treated at the Yonsei University Hospital between January 1985 and December 2001. Patients with granuloma in the pleural biopsy specimen or positive for Mycobacterium tuberculosis in the pleural fluid culture were recruited. The ADA activity in the pleural effusion of 23 renal transplant patients with TPE was compared with 23 immunocompetent patients with TPE. The mean ADA activity was 69.5 +/- 4.6 in renal transplant patients and 65.0 +/- 4.9 U/L in immunocompetent patients. Applying the 40 U/L cut-off point, the positivity of ADA was 91.3% in renal transplant patients, and 86.9% in immunocompetent patients. We thus concluded that the measurement of ADA in the pleural fluid is a useful means in the diagnosis of TPE in renal transplant patients.
Adenosine Deaminase/*metabolism ; Adult ; Female ; Humans ; Immunocompromised Host ; *Kidney Transplantation ; Male ; Pleural Effusion/*diagnosis/*enzymology/microbiology ; Tuberculosis, Pleural/*diagnosis/immunology/metabolism

BACKGROUND/AIMS: The goal of this study was to monitor tuberculosis (TB)-specific T-cell responses in cerebrospinal fluid-mononuclear cells (CSF-MCs) and peripheral blood mononuclear cells (PBMCs) in patients with tuberculous meningitis (TBM) over the course of anti-TB therapy. METHODS: Adult patients (> or = 16 years) with TBM admitted to Asan Medical Center, Seoul, South Korea, were prospectively enrolled between April 2008 and April 2011. Serial blood or CSF samples were collected over the course of the anti-TB therapy, and analyzed using an enzyme-linked immunosorbent spot (ELISPOT) assay. RESULTS: Serial ELISPOT assays were performed on PBMCs from 17 patients (seven definite, four probable, and six possible TBM) and CSF-MC from nine patients (all definite TBM). The median number of interferon-gamma (IFN-gamma)-producing T-cells steadily increased during the first 6 months after commencement of anti-TB therapy in PBMCs. Serial CSF-MC ELISPOT assays revealed significant variability in immune responses during the first 6 weeks of anti-TB therapy, though early increases in CSF-MC ELISPOT results were associated with treatment failure or paradoxical response. CONCLUSIONS: Serial analysis of PBMCs by ELISPOT during the course of treatment was ineffective for predicting clinical response. However, increases in TB-specific IFN-gamma-producing T-cells in CSF-MC during the early phase of anti-TB therapy may be predictive of clinical failure.
Adult ; Antitubercular Agents/therapeutic use ; Biological Markers/blood/cerebrospinal fluid ; *Enzyme-Linked Immunospot Assay ; Female ; Humans ; Interferon-gamma/blood/cerebrospinal fluid ; *Interferon-gamma Release Tests ; Kinetics ; Male ; Middle Aged ; Predictive Value of Tests ; Prospective Studies ; Republic of Korea ; T-Lymphocytes/drug effects/*immunology/metabolism/microbiology ; Treatment Outcome ; Tuberculosis, Meningeal/blood/cerebrospinal fluid/*diagnosis/drug therapy/immunology/microbiology

BACKGROUND/AIMS: Individuals being treated with tumor necrosis factor (TNF)-alpha inhibitors are at increased risk of developing tuberculosis (TB). We determined the clinical characteristics and treatment response of patients who developed TB after using TNF-alpha inhibitors. METHODS: Patients with TB detected within 12 months of the initiation of TNF-alpha inhibitor treatment were included, if seen from January 1, 2000 to August 31, 2011. We retrospectively reviewed the clinical records, results of bacteriological examinations, and radiographs of the included patients and the response to anti-TB treatment. RESULTS: We indentified seven cases of TB in 457 patients treated with TNF-alpha inhibitors during the study period. TB developed a median of 123 days (range, 48 to 331) after the first dose of TNF-alpha inhibitor. Pulmonary TB, including TB pleuritis, was diagnosed in three patients and extrapulmonary TB in four. Favorable treatment outcomes were achieved in six of seven patients. CONCLUSIONS: Among the TNF-alpha inhibitor users who contracted TB, extrapulmonary sites were common and the treatment response was satisfactory.
Adult ; Aged ; Antitubercular Agents/therapeutic use ; Female ; Humans ; *Immunocompromised Host ; Immunosuppressive Agents/*adverse effects ; Interferon-gamma Release Tests ; Male ; Middle Aged ; Retrospective Studies ; Risk Factors ; Time Factors ; Treatment Outcome ; Tuberculin Test ; Tuberculosis/diagnosis/drug therapy/*immunology/microbiology ; Tumor Necrosis Factor-alpha/*antagonists & inhibitors