Low Yield of Chest Radiography in General Inpatients and Outpatients with "Positive PPD" Results in a Country with Low Prevalence of TB.

RATIONALE AND OBJECTIVES: The purpose of this study was to assess the frequency and spectrum of abnormalities on routine screening chest radiographs among inpatients and outpatients with "positive purified protein derivative (PPD)" in a large tertiary care academic medical center in a country with low prevalence of tuberculosis (TB). MATERIALS AND METHODS: The reports of all chest radiographs of general inpatients and outpatients referred for positive PPD (2010-2014) were evaluated for the frequency of evidence of active or latent TB and the spectrum of imaging findings. The results of additional chest radiographs and computed tomography scans were recorded, as were additional relevant clinical histories and symptoms. RESULTS: Of the 2518 patients who underwent chest radiography for positive PPD, the radiographs were normal in 91.3%. The vast majority of the abnormal radiographs demonstrated findings consistent with old tuberculous disease. There were three cases (0.1%) of active TB, all of which were either recent immigrants from an endemic area or had other relevant histories or clinical symptoms suggestive of the disease. CONCLUSIONS: Universal chest radiography in general inpatient and outpatient populations referred for positive PPD is of low yield for detecting active disease in a country with low prevalence of TB.

Comparison of Digital Chest Radiography to Purified Protein Derivative for Screening of Tuberculosis in Newly Admitted Inmates.

This study compares purified protein derivative (PPD) screening to digital chest radiography (CXR) screening for tuberculosis (TB) in newly admitted inmates in the San Diego County Jail system. The study period lasted from 2002 to 2014, during which 45 cases of active TB were detected, a rate of 69.2 cases per 100,000 person-years. Compared to PPD, CXR reduces the median number of days active TB cases were in the general population from 44.4 to 5.2 days and the number of exposures from 1,222 to 138 persons. These results confirm that CXR remains a more effective method for preventing exposure to active TB in jail facilities.

Sarcoidosis and Purified Protein Derivative reactivity.

BACKGROUND: The possible association between (tuberculous and nontuberculous) mycobacterial infections and sarcoidosis is still a matter of dispute. Using diagnostic tests for specific T-cell responses, this association can be investigated in an innovative manner. OBJECTIVE: To measure the T-cell responsiveness to the purified protein derivative (PPD) antigen in blood and broncho-alveolar lavage (BAL) fluid in patients with sarcoidosis and patients with other causes of interstitial lung disease. It was hypothesized that if a mycobacterial infection of the lung is of importance for the development of sarcoidosis, T-cell responsiveness towards the PPD antigen would be increased in patients with sarcoidosis when compared to patients with other causes of interstitial lung disease. METHODS: A single-center study was conducted which included patients with and without sarcoidosis. Venous blood was collected and BAL was performed for, inter alia, Interferon Gamma Release Assay´s (IGRA) with different stimulating antigens, including PPD, ESAT-6, CFP-10 and, as a control, Epstein-Barr virus (EBV). RESULTS: A total of 118 patients were included. There is no difference between PPD reactivity in BAL fluid in patients with or without sarcoidosis. In patients without sarcoidosis, ELISpot PPD in blood shows more reactivity compared to patients with sarcoidosis, although this difference is not significant. ELISpot EBV and TB results are not significant different between both groups. CONCLUSION: These results provide no evidence for the involvement of different mycobacteria in the pathogenesis of sarcoidosis.

Evaluation of gamma interferon and antibody tuberculosis tests in alpacas.

We describe the performance of cell-based and antibody blood tests for the antemortem diagnosis of tuberculosis (TB) in South American camelids (SAC). The sensitivity and specificity of the gamma interferon (IFN-γ) release assay, two lateral flow rapid antibody tests (Stat-Pak and Dual Path Platform [DPP]), and two enzyme-linked immunosorbent assay (ELISA)-based antibody tests (Idexx and Enferplex) were determined using diseased alpacas from Mycobacterium bovis culture-confirmed breakdown herds and TB-free alpacas from geographical areas with no history of bovine TB, respectively. Our results show that while the sensitivities of the IFN-γ and antibody tests were similar (range of 57.7% to 66.7%), the specificity of the IFN-γ test (89.1%) was lower than those of any of the antibody tests (range of 96.4% to 97.4%). This lower specificity of the IFN-γ test was at least in part due to undisclosed Mycobacterium microti infection in the TB-free cohort, which stimulates a positive purified protein derivative (PPD) response. The sensitivity of infection detection could be increased by combining two antibody tests, but even the use of all four antibody tests failed to detect all diseased alpacas. These antibody-negative alpacas were IFN-γ positive. We found that the maximum sensitivity could be achieved only by the combination of the IFN-γ test with two antibody tests in a "test package," although this resulted in decreased specificity. The data from this evaluation of tests with defined sensitivity and specificity provide potential options for antemortem screening of SAC for TB in herd breakdown situations and could also find application in movement testing and tracing investigations.

[Development and use of a domestic test system for diagnosis of tuberculous infection on the basis of quantitative analysis of interferon-gamma induction in whole blood samples in vitro, by using specific recombinant antigens].

A test system was developed to detect tuberculous infection by qualitative analysis of interferon-gamma (IFN-gamma) in the plasma samples after 20-24-hour incubation of whole blood samples in the presence of Mycobacterium tuberculosis (MBT) antigens: tuberculin PPD and a mixture of the MBT-specific recombinant antigens ESAT-6 and CFP-10. The analysis used 3 test tubes each containing 1 ml of heparinized venous blood, one of which served as a control; the other two test tubes were employed to measure antigen-induced IFN-gamma production. Whether this test system might be used to determine primary tuberculous infection was studied in 277 children and adolescents. The threshold diagnostic IFN-gamma induction level determined in the test tube containing a mixture of the antigens ESAT-6 and CFP-10 was ascertained. Postvaccine allergy was detectable if there was IFN-gamma induction in the test tube containing tuberculin and if there was no diagnostic IFN-gamma level in that containing the antigens ESAT-6 and CFP-10. The diagnostic sensitivity of detection of primary tuberculous infection was 97.6% with 94.4% specificity, which enabled this condition to be differentiated from postvaccine allergy. The level of antigen-induced IFN-gamma may be lower in relatively disseminated forms of pulmonary tuberculosis.

Advances in ante-mortem diagnosis of tuberculosis in cattle.

The tuberculin skin test is effective in the early detection of pre-clinical cases of Mycobacterium bovis infection in cattle. This allows the rapid removal of infected animals, thus limiting transmission of the disease, and has resulted in the eradication of bovine tuberculosis (Tb) from many countries. This test is very likely to remain the primary screening test for M. bovis infection in cattle as it is a simple, robust and inexpensive test. However, a number of ancillary tests are being used, or are currently being validated. These ancillary tests are likely to provide a more accurate diagnosis following skin-testing. The blood-based BOVIGAM interferon-gamma (IFN-gamma) test is a cellular immune assay which can detect early infection, and has become the main ancillary test in New Zealand. It can be used for re-testing skin test-positive animals, to improve specificity and minimise wastage from slaughtering animals with false-positive tests. Alternatively, it can be used in locations of increased risk of infection in parallel with skin-testing, for examining skin test-negative animals for pre-movement testing or in problem herds to identify M. bovis-infected animals that do not respond to the skin test. Several modifications of the test are now being used to improve specificity by altering the cut-off or using specific antigens present in virulent mycobacteria such as the 6 kDa early secreted antigenic target (ESAT-6) and 10 kDa culture filtrate protein (CFP-10). While antibody based tests generally lack sensitivity, as high levels of antibodies tend to occur late in the disease process, they may have unique desirable properties such as the ability to be used as a cow-side test. The use of these new ancillary tests in association with skin-testing will improve the detection of M. bovis-infected cattle and reduce the unnecessary slaughter of false-positive reactors.

Is the gamma interferon assay in cattle influenced by multiple tuberculin injections?

Along with other developed countries, Canada is interested in adopting the gamma interferon (IFN-gamma) assay to test for bovine tuberculosis (TB). This study compared results of using the IFN-gamma assay in a large number of field-tested cattle in Manitoba, some previously tested with a caudal fold test (CFT) only, and others injected with tuberculins for both a CFT and a comparative cervical test (CCT). Parallel testing further compared the IFN-gamma assay and CCT results with the confirmed TB status of the animal (culture, histopathologic examination, polymerase chain reaction). Results from IFN-gamma assays did not differ following the CFT versus CFT and CCT injections. Parallel testing demonstrated an apparent higher prevalence of tuberculosis for the IFN-gamma assay versus CCT, which will assist in earlier removal of exposed animals and, ultimately, prevent populations from becoming infected.

The role of flow cytometry in the interferon-gamma-based diagnosis of active tuberculosis and its coinfection with HIV-1--A technically oriented review.

TB remains uncontrolled. In resource-rich countries, only approximately 60% of diagnoses are confirmed by culture. The number is lower in resource-poor environments. Huge scope therefore exists for alternative diagnostic strategies. Counting antigen-specific lymphocytes by virtue of cytokine production following 8-16 h stimulation with tuberculosis antigens is currently the strategy of choice. Several methods exist, including ELISA, ELISpots, and flow cytometry. Although it is clear that blood samples stimulated by ESAT-6 and CFP-10 antigens discriminate between TB infection and BCG vaccination, it is flow-cytometry that seems to be able to distinguish active TB disease from mere TB exposure. Of the various flow-protocols including four-color tests (CD45-CD3-CD4-IFNgamma), three-color tests (CD3-CD4-IFNgamma) and two-color tests (CD4-IFNgamma), even the simplest is performing well, provided that the results are expressed as percentage of IFN-gamma+ cells per CD4+ lymphocytes (%IFNgamma/CD4+). Studies using broncho-alveloar lavage (BAL) and Induced-Sputum (ISp) show that TB-specific CD4+IFN-gamma+ T cells accumulate in the lung in pulmonary and extra-pulmonary TB at frequencies >5-20-fold more frequent than in blood. This pulmonary homing is absent following BCG immunization. The use of PPD to stimulate CD4+IFN-gamma+ cells in the lung in active TB leads to >3-12-fold greater responses than seen with CFP-10 or ESAT-6, and any interference from BCG vaccination is absent. This method is unaffected by HIV coinfection, which has always been the problem for other immune-based diagnostics. Further, lung-based samples provide material for rapid tests of both the IFN-gamma assay and bacteriology, and importantly, these tests are amenable for future simplification with automated fluorescence-image cytometers.Another development of the multiparameter analytical power of flow-cytometry is to use markers for "lung-seeking" populations of CD4+ T cells in blood, obviating lung sampling. In active TB, but not in BCG vaccinees, TB-specific memory CD4+ T cells can be found in blood that are dominantly CD27-negative and probably lung seeking and can be diagnostically useful.

Genome-wide scan for loci influencing quantitative immune response traits in the Belém family study: comparison of methods and summary of results.

Here we report the results from a genome-wide linkage scan to identify genes and chromosomal regions that influence quantitative immune response traits, using multi-case leprosy and tuberculosis families from north-eastern Brazil. Total plasma IgE, antigen-specific IgG to Mycobacterium leprae soluble antigen (MLSA), M. tuberculosis soluble antigen (MTSA) and M. tuberculosis purified protein derivative (PPD), and antigen-specific lymphocyte proliferation (stimulation index or SI) and interferon-gamma (IFN-gamma) release to MLSA and PPD, were measured in 16 tuberculosis (184 individuals) and 21 leprosy (177 individuals) families. The individuals were genotyped at 382 autosomal microsatellite markers across the genome. The adjusted immune-response phenotypes were analysed using a variety of variance components and regression-based methods. These analyses highlighted a number of practical issues and problems with regard to implementation of the methods and, interestingly, differences were observed between several standard statistical and genetic analysis packages used. From this we determined that, for this set of traits in these pedigrees, significant p values for linkage using variance components analysis, supported by significance using the Visscher-Hopper modification of the Haseman-Elston method, provided the most compelling evidence for linkage. Using these criteria, linkage (5.8 x 10(-5) < p < 0.008) was seen for: total plasma IgE on chromosome 2; IgG to MLSA on chromosomes 8, 17 and 21; IgG to PPD on chromosome 12; SI to PPD on chromosome 1; IFN-gamma to MLSA on chromosomes 6, 7, 10, 12 and 14; and IFN-gamma to PPD on chromosomes 1, 16 and 19.

Comparison of purified protein derivatives and effect of skin testing on results of a commercial gamma interferon assay for diagnosis of tuberculosis in cattle.

Purified protein derivatives (PPD) prepared in the USA were compared with those prepared in Australia by a private company (CSL Veterinary) for use with a commercial gamma interferon (gamma-IFN) assay for diagnosis of bovine tuberculosis. The effect of skin testing on results of the gamma-IFN assay was determined, and results were compared when blood samples were stimulated with PPD within 2 hours and after 24 hours of sample collection. Twenty cattle that were sensitized by subcutaneous injection of heat-killed Mycobacterium bovis were randomly divided into 3 groups. Cattle in group A were tested with the caudal fold skin test (CFT) on day 0 and the comparative cervical skin test (CCT) on day 7. Cattle in group B were tested with the CFT on day 0 and the CCT on day 63, and group C cattle were not skin tested. Blood samples for the gamma-IFN assay were collected at various times throughout the study period. Optical density (OD) values for the gamma-IFN assay were not significantly different when blood samples were stimulated with US avian PPD and CSL avian PPD. However, OD values were significantly higher for US bovine PPD than for CSL bovine PPD. However, the final interpretation of the gamma-IFN assay was usually the same when using either US or CSL PPD. In addition, OD values for the gamma-IFN assay were significantly higher for blood samples collected after sensitized cattle were skin tested than for samples collected from the same cattle before skin testing or from cattle not skin tested. The OD values for blood samples stimulated within 2 hours of sample collection were significantly higher than for samples stimulated 24 hours after sample collection. However, OD values for all PPD-stimulated samples from sensitized cattle were significantly higher in samples collected 3 days after skin testing and stimulated 24 hours after collection than for samples from the same animals collected before skin testing and stimulated within 2 hours of sample collection. Results of this study indicate that PPD prepared in the USA or Australia can be used to stimulate blood samples for the gamma-IFN assay. Skin testing cattle prior to collection of blood for the gamma-IFN assay boosts production of gamma-IFN by lymphocytes from cattle that have had prior exposure to M. bovis antigens. Use of the gamma-IFN assay in conjunction with skin testing may improve detection of cattle infected with M. bovis. In addition, the increase in production of gamma-IFN after skin testing will permit greater flexibility in conducting the assay because samples can be stimulated after they have been shipped overnight rather than only on the day of sample collection.

Classically restricted human CD8+ T lymphocytes derived from Mycobacterium tuberculosis-infected cells: definition of antigenic specificity.

Previous studies in murine and human models have suggested an important role for HLA Ia-restricted CD8(+) T cells in host defense to Mycobacterium tuberculosis (Mtb). Therefore, understanding the Ags presented via HLA-Ia will be important in understanding the host response to Mtb and in rational vaccine design. We have used monocyte-derived dendritic cells in a limiting dilution analysis to generate Mtb-specific CD8(+) T cells. Two HLA-Ia-restricted CD8(+) T cell clones derived by this method were selected for detailed analysis. One was HLA-B44 restricted, and the other was HLA-B14 restricted. Both were found to react with Mtb-infected, but not bacillus Calmette-Guérin-infected, targets. For both these clones, the Ag was identified as culture filtrate protein 10 (CFP10)/Mtb11, a 10.8-kDa protein not expressed by bacillus Calmette-Guérin. Both clones were inhibited by the anti-class I Ab and anti-HLA-B,C Abs. Using a panel of CFP10/Mtb11-derived 15-aa peptides overlapping by 11 aa, the region containing the epitopes for both clones has been defined. Minimal 10-aa epitopes were defined for both clones. CD8(+) effector cells specific for these two epitopes are present at high frequency in the circulating pool. Moreover, the CD8(+) T cell response to CFP10/Mtb11 can be largely accounted for by the two epitopes defined herein, suggesting that this is the immunodominant response for this purified protein derivative-positive donor. This study represents the first time CD8(+) T cells generated against Mtb-infected APC have been used to elucidate an Mtb-specific CD8(+) T cell Ag.

Analysis of circulating immune complexes (CICs) in childhood tuberculosis: levels of specific antibodies to glycolipid antigens and relationship with serum antibodies.

BACKGROUND: The presence of specific antiglycolipid antibodies in serum and circulating immune complexes (CIC) in children with tuberculosis was detected in order to evaluate their contribution to the value of serodiagnosis of tuberculosis, as has already been shown in adults. METHODS: ELISAs using the three glycolipids LOS, DAT and PGLTb1 were performed in whole serum and immune complexes from 20 children with tuberculous disease or infection, in seven child contacts, and in 26 children with non-tuberculous disease. The contribution of complexed IgG antibody to the diagnostic values was established for each group. RESULTS: The antibody levels in free serum were higher (P < 0.01) in children with tuberculous disease or infection and in contacts than in controls. By contrast, except for PGLTb1, the IgG antibody levels were higher (P < 0.02) in children with tuberculous disease than in the other groups. The highest contribution of IgG antibody against LOS to the predictive values was shown in children with pulmonary tuberculosis (positive predictive value 1,000, negative predictive value 1,000). In paucibacillary tuberculosis (extra-pulmonary and tuberculous infection) and in contacts, the IgG antibody did not contribute to the sensitivity of the serodiagnosis, where the combination of antigens tested in serum increased the diagnostic yield. The very low levels of IgG antibody in these settings may indicate a different B cell response. CONCLUSION: The detection of immune complexes and IgG antibodies against the three glycolipid antigens is useful as a complementary technique for the serodiagnosis of children with a high probability of pulmonary tuberculosis.

Activation of human CD8+ alpha beta TCR+ cells by Mycobacterium tuberculosis via an alternate class I MHC antigen-processing pathway.

Human immune responses to M. tuberculosis are characterized by activation of multiple T cell subsets including CD4+, CD8+, and gammadelta T cells, and the role of CD8+ alphabeta TCR+ T cells in this response is poorly understood. Stimulation of T cells from healthy tuberculin skin test-positive persons with live M. tuberculosis-H37Ra or soluble M. tuberculosis Ags readily up-regulated IL-2Ralpha (CD25) expression on CD8+ T cells. Purified resting and activated CD8+ T cells produced IFN-gamma and proliferated to both M. tuberculosis bacilli and soluble mycobacterial Ags with monocytes as APC. Precursor frequency of mycobacterial Ag-specific CD8+ T cells by IFN-gamma enzyme-linked immunospot was 5-10-fold lower than the precursor frequency of CD4+ T cells, and IFN-gamma secretion by CD8+ T cells was 50-100-fold lower. CD8+ T cells secreted approximately 10-fold less IFN-gamma per cell than CD4+ T cells in response to mycobacterial Ags. CD8+ T cell responses to M. tuberculosis bacilli were blocked by anti-MHC class I antibody and required Ag processing. Processing of M. tuberculosis bacilli by monocytes for presentation to MHC class I-restricted CD8+ T cells was insensitive to brefeldin A treatment, which blocks the conventional MHC class I Ag-processing pathway. These results represent the first demonstration that human cells can process pathogen Ags via an alternate Ag-processing pathway for MHC class I and suggest a mechanism for participation of IFN-gamma-secreting CD8+ T cells in the human immune responses to M. tuberculosis.

IL-13 over-expression in skin is not confined to IgE-mediated skin inflammation.

IL-13 is produced by T cells and, like IL-4, it can induce the production of IgE and IgG4. In order to investigate if IL-13 is a specific marker for atopic dermatitis (AD), IL-13 gene expression was analysed in chronic lichenified lesions and non-lesional skin of patients with AD, in involved and non-involved skin of patients with psoriasis, in positive tuberculin reactions in non-atopics, and in the skin of healthy control subjects. Patients with AD (n = 9) showed sensitization to common air-borne allergens (positive Phadiatop) and had total serum IgE values in the range from 10 to 4800 kU/l (median 170 kU/l). Competitive reverse transcription-polymerase chain reaction (RT-PCR) was used to assess IL-13 gene expression in skin biopsy specimens. IL-13 gene expression was markedly higher in chronic lichenified lesions of patients with AD (P < 0.01), and in the positive tuberculin reactions (P < 0.01; n = 12) than in skin from healthy control subjects (n = 10). However, there was no significant difference in IL-13 gene expression in the skin of patients with psoriasis (n = 10) and that of healthy control subjects. The dermal cell infiltrates were larger and the relative amount of CD3+ and CD4+ cells in these infiltrates was higher in the skin of subjects with a positive tuberculin reaction than in lichenified AD skin. However, these differences were not reflected in differences in IL-13 gene expression. Different triggers of IL-13 gene expression may influence the diverse patterns of inflammation seen in different inflammatory skin disorders.

[Use of immunochemical studies to predict the course of fibrous cavernous tuberculosis of lung and postoperative complications in patients on chemo and laser therapy]. / Ispol'zovanie immunokhimicheskikh issledovanií dlia prognoza techeniia fibrozno-kavernoznogo tuberkuleza i posleoperatsionnykh oslozhnenií u bol'nykh, poluchavshikh khimio- i lazerterapiiu.

A total of 103 patients with fibrocavernous tuberculosis of the lung were examined. They all received chemotherapy, including 3 - 4 antituberculous agents. Laser therapy was performed with a UZOR-2K low-energy semiconductor laser. In patients with profound changes in the serum level of protein, with high antigenemia and antibody production, the course of the disease was found to be poor; X-ray positive changes were achieved to a lesser extent, bacterial expellation stopped less frequently and more slowly. The decreases in the serum content of the proteins tested, in the level of antigenemia and antibody production which occur with drug and laser therapies are also an important factor of preoperative preparation, which is highly effective in preventing postoperative complications.

Serological diagnosis of tuberculosis.

The past decade has seen dramatic developments in serological tests for tuberculosis. The long history of serological tests for tuberculosis is a testimony to the need for a sensitive diagnostic test, especially when the sputum smear is unhelpful. New reagents, both purified antigens and monoclonal antibodies, provide the means to obtain a sensitivity and specificity to rival the tuberculin skin test and equal other commonly used diagnostic blood tests. Evaluation with sera from patients with smear-positive pulmonary tuberculosis has identified one antigen (antigen 5, the 38 kDa antigen) as a potential screening reagent for infectious tuberculosis and another (16 kDa antigen) for monitoring compliance. A monoclonal antibody competition assay anti-38 kDa antibody is the most sensitive serological test for smear-negative tuberculosis so far. Tests for tuberculous meningitis need clinical evaluation. Serological tests for human immunodeficiency virus (HIV)-related tuberculosis are disappointing. In general, antibody levels in primary tuberculosis are low and appear directed towards cytoplasmic antigens, whilst in post-primary disease antibody levels are higher and appear to bind to secreted antigens. No single reagent gives a 100% sensitivity; future research should identify the best combination of antigens for the serodiagnosis of tuberculosis.