Thin layer chromatography characterization of ELISA specific glycolipids antigens immunomagnetically purified from environmental mycobacteria.

Thin layer chromatography (TLC) could easily and rapidly evidentiate the qualitative differences between glycolipids (GLs). Different immunomagnetically purified mycobacterial GLs have been compared using TLC, in order to choose the most appropriate antigens to be utilized in ELISA. The GLs were purified from environmental mycobacteria (EM) (M. avium-intracellulare, M. kansasii, M. xenopi, M. scrofulaceum and M. gordonae) and from M. tuberculosis H37Rv. BioMag Amine and BioMag Carboxyl terminated superparamagnetic microparticles were utilized in the magnetic separation of glycolipids from mycobacterial species. TLC of GLs before and after magnetic purification, corroborated with ELISA results, shows that COOH-terminated particles allow a better purification for M. kansasii, M. xenopi and M. scrofulaceum, while NH2-terminated particles act better on MAI and M. gordonae GLs. The use of GL purified antigens in ELISA could fulfill the criteria of high levels of both sensitivity and specificity of serologic assays in EM diagnosis.

Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

BACKGROUND & OBJECTIVES: Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. METHODS: Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. RESULTS: For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 µg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. INTERPRETATION & CONCLUSIONS: Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

Serodiagnosis of environmental mycobacterial infections.

To demonstrate the usefulness of enzyme-linked immunosorbent assay for serodiagnosis of mycobacterioses due to environmental mycobacteria we utilized a panel of glycolipid antigens selective for Mycobacterium avium-intracellulare, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium scrofulaceum and Mycobacterium gordonae. The levels of circulating antibodies were determined against the environmental mycobacteria, and Mycobacterium tuberculosis in human immunodeficiency virus-negative and -positive patient sera. The method used immunomagnetic separation of the antigens, with covalent immobilization of antibodies to superparamagnetic amine and carboxyl terminated particles in solutions of the specific antigens. Enzyme-linked immunosorbent assay was performed on 195 patient sera: 34 with infections due to environmental mycobacteria, 114 with tuberculosis, 47 with other respiratory diseases. There were 46 human immunodeficiency virus-1 infected individuals. Among the 34 infections due to environmental mycobacteria, 9 patients were singularly infected with an environmental mycobacterium, and 25 co-infected with both M. tuberculosis and an environmental mycobacterium. Sensitivity, specificity and false positivity ranges were determined for each of the volunteer groups: tuberculosis positive, human immunodeficiency virus negative; tuberculosis positive, human immunodeficiency virus positive; those with infections due to individual environmental mycobacteria (such as M. scrofulaceum and M. kansasii); and those with other respiratory diseases. We demonstrate that such multiple assays, can be useful for the early diagnosis of diverse environmental mycobacterial infections to allow the start of treatment earlier than henceforth.

Comparative study of RT23 and IC-65 tuberculins tested on children with tuberculosis.

This study, conducted in 2009, proposed to evaluate and compare the biological potency of two different tuberculins, RT23 (Statens Serum Institute, Copenhagen) and IC-65 (Cantacuzino Institute, Bucharest) when administered to 89 children with confirmed tuberculosis, admitted to Paediatric Department of Pneumophtysiology Institute, Bucharest. Mean age of subjects was 10.4 years [SD (standard deviation) = 5.2 years; variance = 27.2], and sex distribution in the group was: 55.1% girls and 44.9% boys. Tuberculin skin tests were performed using Mantoux method simultaneously with the two tuberculins in the same concentration, 2TU (tuberculin units)/0.1 ml. RT23 skin test reactions ranged from 8 mm to 18 mm (mean = 12.8 mm, SD = 2.1 mm, variance = 4.4; median = 12.0), and IC-65 reactions ranged from 8 mm to 18 mm (mean = 13.1 mm; SD = 2.1 mm; variance = 4.3; median = 13.0). The mean difference in paired reaction sizes for the two reagents was 0.04 mm and was not statistically different from zero (P value = 0.3). The difference in reaction sizes was = 2 mm in 70.8% and = 5 mm in 7.9% patients. With a cutoff of 10 mm to define a positive reaction, the results were highly correlated with a sensitivity of 98.9% for RT23 and 97.8% for IC-65. No statistically significant difference was established for the efficacy of the two commercially available PPD TST reagents, both tuberculins appearing to have equivalent potency.

Prospective Comparison of Two Brands of Tuberculin Skin Tests and Quantiferon-TB Gold in-tube Assay Performances for Tuberculosis Infection in Hospitalized Children.

OBJECTIVE: The study compared two brands of tuberculin skin tests (TST): PPD RT 23, (SSI, Denmark) and PPD IC 65, (Cantacuzino Institute, Romania), 2 TU/ 0.1 ml each, with an interferon gamma release assay [IGRA], Quantiferon-TB Gold (QFT). MATERIAL AND METHODS: QFT was performed on whole blood samples, before TSTs, on 60 children with tuberculosis (TB), BCG vaccinated, admitted in a paediatric pneumophtisiology hospital. The proportion of boys (51.6 %) and girls (48.3 %) was nearly equal, the mean age of subjects was 9.44 years (SD= 5.37 years; variance= 28.83). RESULTS: With TST induration ≥ 10 mm considered as positive response, only 47.46 % of children classified positive with RT23 and 48.27 % with IC-65, were IFN-γ positive.We obtained a very good agreement between the two tuberculins (59/60 for RT 23 and 58/60 for IC 65), while for QFT, which confirmed as positives only 27/60, i.e. 45 % (18/60 were indetermined, 15/60 were negatives). CONCLUSIONS: The tests did not agree on positive results, showing a low redundancy between in vitro and in vivo measurements, suggesting that independent aspects of anti-mycobacterial immunity are being measured by these tests.The specificities of the assays could not been calculated since all the children had TB, confirmed by bacteriological and/ or clinical and radiological data. Further comparison of TST and QFT, may determine whether such discordance reflect a higher specificity of QFT. Meantime, we are trying to obtain a recombinant PPD, using a cocktail of specific M. tuberculosis (M.tb) antigens, in order to eliminate any interference with BCG in skin test reactions.

Comparison of tuberculin skin test with a whole-blood interferon gamma assay and ELISA, in HIV positive children and adolescents with TB.

We compared the usefulness of three methods designed to diagnose latent tuberculosis [TB]: interferon-gamma release assay [IGRA], such as QuantiFERON-TB Gold [QFT-G], Enzyme-linked immunosorbent assay [ELISA] serologic assay and tuberculin skin test [TST] for diagnosis of TB in human immunodeficiency virus [HIV]-1 infected children and adolescents, with microbiologically and/or histopathologically confirmed TB co-infection. The serum samples were obtained from 36 patients who were examined and tested by the three methods. The sensitivity was 38.8% for TST, 47.2% for IGRA (QFT-G) and 11.1% for ELISA. Out of 24 patients with severe immune suppression (CD4+ < 200 cells/ml), 6 had positive TST, i.e. sensitivity 25%, 10 positive QFT-G results, i.e. sensitivity 41.6%. 6 of the QFT-G results could not been determined. ELISA was positive only for one of them. Among the 12 patients without severe immune suppression (CD4+ > 200 cells/ml), 8 had positive TST, QFT-G was positive in 7 patients., 3 of QFT-G results could not been determined. ELISA was positive in 3/12 patients. Only 3 of these results were simultaneously positive with TST, QFT-G and ELISA. Our results demonstrated concordance between QFT-G and TST in HIV-infected children and adolescents diagnosed with TB. Since all the patients had active TB, it was not possible to calculate the specificity of the tests. ELISA had the lowest sensitivity, while QFT-G and TST sensitivities were comparable for the children and adolescents with CD4+ count >200 cells/ml. Further research is needed in HIV-1 positive children and adolescents with and without TB in order to validate rapid diagnosis methods for TB.