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.

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.

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.

Rapid identification of Mycobacterium species by lectin agglutination.

The purpose of the present study is to explore the possibility that plant lectins can be used for the development of rapid and inexpensive technique for differentiation of mycobacterial species. The method is based on interaction between mycobacteria and lectins as visualized by agglutination in a microtiter plate. We employed 18 mycobacterium species and determined the minimal lectin concentration (MLC) of 23 different lectins. For some of the bacteria as a high as 1000 microg/ml of one or more lectins were required to induce agglutination, while for other strains as low as 1.95 microg/ml of the lectin were needed. A unique pattern of agglutination was observed for each species over a range of 62-1000 microg/ml lectin concentrations. There were little or no variations in MLC within strains (intraspecies) of each of two species tested. In contrast, there were marked interspecies variations in MLC. Analysis of the MLC showed that the highest score of interspecies differences with 23 lectins was obtained at 125 microg/ml lectin concentration. At this concentration it was found that the pattern of agglutinations with only two lectins was sufficient to differentiate mycobacterium species from each other. Because the bacteria-lectin interaction is adaptable to various methods of visualization, our findings may set the stage for developing a rapid and reliable tool to differentiate mycobacterium species.

Establishing of a minimal set of tests (minimal standards) to identify species included in the genus Mycobacterium.

Mycobacterium genus includes over 100 species and subspecies; new species are discovered every year. Minimal standard criteria are represented by the resistance to acid-alcohol (e.g. in the Ziehl - Neelsen staining), the presence of some mycolic acids containing 60-90 carbon atoms that can be cleaved by pyrolysis in fatty acids with 22 - 26 carbon atoms and a guanine + cytosine content of the DNA of 61 to 71 mol %. The species with the highest rate of involvement are those from Mycobacterium tuberculosis complex, and tuberculosis is still one of the most widespread world diseases. The most important for a laboratory is to be able to identify the species from M. tuberculosis complex. We have done a series of experiments, their goal being to evaluate and establish a minimal set of useful tests for identification of mycobacterial species. We used strains from "Cantacuzino" Institute collection and applied a series of classical and modern methods. We appreciate that the minimal set of tests could be represented by the microscopic examination for acid-fast bacilli (AFB), the examination for the preferred growth temperature, the growth rate, the colonies morphology, pigmentation and photo reactivity, the niacin accumulation test, the test of nitrate reduction, the catalase test (in both variants), plus the susceptibility to Para-Amino Salicylic Acid, Para-Nitro-Benzoic Acid andto Tiophene-2-Carboxylic Acid Hydrazide.

Rapid immunochromatographic serum assay of nontuberculous mycobacterial infections.

A rapid immunochromatographic serologic assay (Dot assay) is proposed to be applied on patients infected with nontuberculous mycobacteria (NTM). This assay could evidentiate the infecting species and allow the beginning of the treatment. The test is based on the principle of immunoblotting chromatography, a rapid membrane-based assay, capable of diagnosing NTM infections in serum, in less than 1 hour, with no need of special equipment or skilled staff. The secreted extracellular antigens have been isolated from the unheated culture filtrates of the clinically significant NTM (M. avium, MAI, M. kansasii, M. xenopi, M. chelonaei, M. scrofulaceum, M. marinum, M. fortuitum, M. abscesus, M. szulgai). The patients have been tested against these antigens, as well as from M. tuberculosis H37Rv, due to the possibility of co-infection with tuberculous bacilli. A number of 385 tests on patient sera have been performed (10, with NTM suspected infection, with or without M. tuberculosis co-infection, 5 with confirmed diagnosis of NTM infection, 10 with TB, 10 with other respiratory diseases). The preliminary results presented in this paper support the fact that the rapid immunochromatographic serum assay, combined with clinical and radiographic evidence, could evidentiate the infecting NTM species and allow the start of an earlier treatment, but must be confirmed on a higher number of patients.

Rapid dot sputum and serum assay in pulmonary tuberculosis.

A rapid direct sputum (Sp.) and/or antibody assay, based on immunoblotting and enzyme immunoassay is described. The test can detect mycobacterial antigens or antibodies in clinical specimens from pulmonary tuberculosis (TB) patients. In this study, 87 sputa, 87 sera and 40 paired sputa and sera were utilized from smear-positive and smear-negative, culture-positive patients; 59 sputa, 37 sera and 22 paired sputa and sera from nontuberculosis respiratory disease patients and 68 sera from healthy controls. The antigen detection in sputum by dot assay has 86.1% sensitivity on active tuberculosis patients, 92.9% specificity, 91.6% positive predictive value (PPV), 88.2% negative predictive value (NPV) and 10.3% error. The antibody assay has 83.6% sensitivity, 95.4% specificity, 94.4% positive predictive value, 85.6% negative predictive value and 11% error. The test performed on paired sputum and serum (Sr.) samples has a sensitivity of 93.3%, which rose to 96.1% on smear-positive and culture-positive patients, but the specificity decreased to 83% in sputum, whereas in serum it was 92%. The results of the assay, combined with clinical and radiological data, could form the basis for starting an earlier course of treatment for tuberculosis.

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.

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.

Hemagglutinin of unusual specificity from Helcococcus kunzii.

Helcococcus kunzii is a gram-positive, catalase-negative opportunist. The organism has been isolated from the lower extremities and breast masses of several patients. A clinical isolate of Helcococcus kunzii was shown to possess a hemagglutinin-lectin with a specificity for N-acetylglucosamine and lactose, two structurally unrelated carbohydrates. The lectin is sensitive to protease, heat and mutanolysin. Electron microscopy failed to reveal fimbriae or fibrillae, suggesting that the lectin is associated with peptidoglycan or the cytoplasmic membrane. It is likely that the lectin is involved in adhesion and colonization of H. kunzii.