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Objective To investigate the treatment evaluating value of ESAT－6 and CFP－10 in T－SPOT.TB kit for tuberculous vertebral osteomyelitis.Medthods This retrospective study analyzed 29 cases diagnosed as TVO in the First Affiliated Hospital of Kunming Medical University from January 2013 to January 2016. All cases were the first－time consultancy. The Wilcoxon－singed－rank－test and chi－square test were used to analyze the changes of ESAT－6 and CFP－10 in the procedure of treatment. The linear－regression analysis was used to analyze the relationship between ESR, CRP, VAS and two specific antigens.Results The pretherapeutic spot counts of ESAT－6 and CFP－10 were compared with the first and the last follow－up respectively. The results of two antigenic spots change showed a higher consistency (P＜0.05) .The positive rates of CFP－10 at the prior treatment, the first follow－up and the last follow－up were 86.20% , 79.31% and 58.62% respectively. The result of chi－square test showed a higher consistency (P＜0.05) . ESAT－6 only correlated with VAS. CFP－10 had the relationship with VAS and ESR. But all of these relativities were weak.Conclusion The decrease in the spot counts of ESAT－6 and CFP－10 suggest that the treatment is effective,and CFP－10 may be one available index to evaluate the treatment effect.
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Objective To investigate the Mycobacterium tuberculosis infections in 2014 among Beijing army recruits, and evaluate a new method for screening latent tuberculosis infections.Methods A total of 194 army recruits were subjected to chest X-ray examination purified protein derivative(PPD) skin test, antibody detection, and interferon gamma release assay(IGRA) by ELISA combined with recombinant protein CFP10-ESAT6 and latent infection protein Rv2628.Results The positive rates of PPD skin test and antibody test were 49.7% and 15.5%, respectively.The latent infection rate of IGRA test was 22.2% in 194 cases after CFP10-ESAT6 stimulation.After stimulation of latent tuberculosis infection(LTBI) with Rv2628, IFN-γ level was significantly higher than that in healthy control group (P0.05).There was no significant difference between antibody negative and positive groups after stimulation by CFP10-ESAT6 and Rv2628 (P>0.05).The area under the ROC curve of Rv2628 diagnosis of tuberculosis infection was 0.84.When Youden index was 0.621,the specificity was 94.7% and sensitivity was 67.4%.ConclusionCombined detection of antigens Rv2628 and CFP10-ESAT6 specific IFN-γ values can be potentially used for differential diagnosis of active or latent tuberculosis infections.
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Objective To investigate the effect of Shenmai injection on cytokines and ESAT-6/CFP-10 levels in patients with Chronic obstructive pulmonary disease(COPD)with pulmonary tuberculosis.Methods158 patients with COPD with pulmonary tuberculosis from the Department of Respiratory in our hospital were selected and divided into 2 groups, 79 cases in the control group treated with routine treatment, 79 cases in the experiment group treated with Shenmai injection on the basis of the control group, levels of neurotransmitters in the brain, serum ESAT-6 protein, CFP-10 protein, IFN-γ levels, cytokine levels, peripheral blood immune cell levels, and the clinical effect and the focus absorption efficiency were compared after the treatment.ResultsThe clinical total effective rate in the control group(84.81%)was lower than that in the experiment group(94.94%), with significant difference (P<0.05);the focus absorption efficiency in the control group(87.33%)was lower than that in the experiment group(96.21%), with significant difference (P<0.05);compared with the control group, serum ESAT-6 protein、 CFP-10 protein、IFN-γ levels were lower after treatment in the experiment group, the serum levels of TNF-α、IL-6、sIL-2R were lower, peripheral blood levels of NK cells, CD4+T lymphocytes and CD4+/CD8+ were higher, peripheral blood level of CD8+T lymphocytes was lower, with significant difference (P<0.05).ConclusionShenmai injection can significantly reduce the serum levels of TNF-α、IL-6、sIL-2R and ESAT-6/CFP-10 in patients with COPD with pulmonary tuberculosis, improve cellular immune function, promote the absorption of lesions effectively,and improve clinical efficacy.
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Objective To clone and express Mycobacterium tuberculosis fusion antigen ESAT-6 and CFP-10 ( EC) and to evaluate the biological activity of the fusion antigen EC in inducing specific cytokines secretion from THP -1 cells. Methods The fusion antigen EC gene was cloned into pET-30 a prokaryotic expression vector and expressed highly in E.coli BL21.Then, the THP-1 cells were stimulated with purified fusion antigen EC of different concentrations (10 and 20 μg/ml).Culture supernatants were collected after 12 h and 24 h, respectively.The secretion levels of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-αand IFN-γin THP-1 cell culture supernatants were detected using Bio-PlexProTM Assays kit.Results The M.tuberculosis fusion antigen EC was cloned and expressed successfully .The secretion levels of IL-6, IL-8 and TNF-αin EC infected THP-1 cells were significantly higher than those in THP-1 cells (P<0.05).The secretion levels of other cytokines did not change significantly .Conclusion The obtained M.tuberculosis fusion antigen EC has biological activity in inducing the THP-1 cells to secrete a higher level of IL-6,IL-8 and TNF-α.
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The RD-1 locus has been considered crucial in the pathogenesis of M.tuberculosis, the RD-1 locus is 9.5 kb and spanning open reading frames Rv3871 to Rv3879c encoding 9 different proteins separately.The RD-1 locus is missing in all bacillus Calmette-Guerin(BCG) strains, and is one of the key virulence factor in M.tuberculosis.The RD-1 locus participates in a new secreting system named ESX-1, which can facilitate the secretion of some special proteins.The two important proteins encoded by the RD-1 locus named CFP-10 and ESAT-6 can form a tight 1∶1 complex, and has been shown to be coordinately secreted and lead to a strong T cell response, which suggests that these two proteins may act as ideal target antigens in diagnosis and prevention of tuberculosis(TB).
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BACKGROUND:The tuberculin skin test, which has been used for years for the diagnosis of latent tuberculosis, has many limitations, including false-positive results in individuals who were vaccinated with BCG. We evaluated the usefulness of a recently developed interferon-gamma assay (QuantiFERON-TB Gold) in the diagnosis of latent tuberculosis. METHODS:We performed the QuantiFERON-TB Gold assay in the following groups: 1) individuals with negative responses of tuberculin skin test in the regular health checkups for two consecutive years (n = 14), 2) individuals with no abnormal findings in low dose computed tomography in a health checkup (n = 22), 3) individuals with stable tuberculosis in low dose computed tomography in a health checkup (n = 10), 4) patients with M. tuberculosis in culture (n = 23), 5) patients with nontuberculous mycobacteria in culture (n = 6). RESULTS:In the QuantiFERON-TB Gold assay, all the group 1 showed negative results. 65.2% of the group 4 showed positive QuantiFERON-TB Gold results, while all the group 5 showed negative results. 22.7% of the group 2 and 60.0% of the group 3 showed positive QuantiFERON-TB Gold results. In addition, it was revealed that the stimulation with CFP-10 played a major role in the induction of interferon-gamma secretion. CONCLUSION:The QuantiFERON-TB Gold assay shows promise for the immunodiagnosis of latent tuberculosis using a whole-blood.
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Humanos , Diagnóstico , Testes Imunológicos , Interferon gama , Tuberculose Latente , Mycobacterium bovis , Micobactérias não Tuberculosas , Testes Cutâneos , Tuberculina , TuberculoseRESUMO
Objective:To construct lhp-esat6 fusion gene and its prokaryotic expression vector and express it in E.coli.Methods:By Gene SOEing techniques,a fusion gene was constructed by splicing lhp gene and esat6 gene,then cloned into pQE30 plasmid and expressed in DH5a.Results:The fusion gene was identified by DNA sequencing.A fusion protein about 26kDa was expressed in the E.coli.In presence of 1mM IPTG for 4h,the fusion protein was expressed to the maximum.The fusion protein existed in cytoplasm in soluble form and represented about 40% total bacterial protein of E.coli.Its antigenicity was confirmed by Western blotting.The fusion protein was purified through the Ni-NTA resin and the purity reached 98%.Conclusion:The lhp-esat6 fusion gene and the prokaryotic expression vector (pQE30-CFP10-ESAT6) were constructed successfully,and the fusion protein CFP10-ESAT6 was obtained,so that it can provide an experimental basis for application of the recombinant CFP10-ESAT6.