<b><span style="font-family:Verdana;">Background:</span></b><span style="font-family:Verdana;"> In patients with HIV, conventional tests are of low sensitivity;therefo...<b><span style="font-family:Verdana;">Background:</span></b><span style="font-family:Verdana;"> In patients with HIV, conventional tests are of low sensitivity;therefore, a new diagnostic test with hydrogel nanoparticles with reactive blue dye is proposed, which allows the capture, conservation and concentration of ESAT-6 in urine. NIPAs are copolymers that capture low molecular weight proteins and protect against enzymatic degradation. Using an immunoassay, it is possible to detect and quantify ESAT-6 present in urine as a possible marker of active TB. <b></b></span><b><b><span style="font-family:Verdana;">Design/Methods:</span></b><span style="font-family:Verdana;"></span></b> Study in Lima, Peru, HIV+ participants, ≥18 years with and without tuberculosis (TB). Smear microscopy, culture in solid medium and urine immunoassay were performed. The reference was the diagnosis of TB by radiological or clinical microbiological criteria (indication for TB treatment). There were 2 preanalytical processes: untreated and treated urine (centrifuged, heated), then incubation with NIPAm. After washing, elution, sonication, heat and centrifugation, the final eluate was obtained. This was placed on nitrocellulose membranes, which by means of fixation and incubation processes with anti-ESAT-6 and anti-IgG antibodies, revelation with C-DiGit<span style="white-space:nowrap;">®</span> Blot Scanner and FluorChem R FR0001. Calibration curves were included in the membranes, density was measured using Image J software. ROC curves, sensitivity and specificity were obtained. <b><b><span style="font-family:Verdana;">Results:</span></b><span style="font-family:Verdana;"></span></b> The result by groups was HIV+ patient: ROC: 0.75<span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">,</span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">c</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">ut-off point ≥24.06 ng/ml, sensitivity 76.32%, specificity 68.89%, patients ≤200 cells CD4 mm<sup>3</sup>/ml, ROC: 0.78, cutoff point ≥26.20 ng/ml, sensitivity 75.86%, specificity 71.88%, patients</span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span><span><span style="font-family:;" "=""><span style="font-family:Verdana;">>200 CD4 mm</span><sup><span style="font-family:Verdana;">3</span></sup><span style="font-family:Verdana;"> cells/ml, ROC</span></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">:</span></span></span><span><span><span style="font-family:;" "=""><span style="font-family:Verdana;"> 0.73, cutoff point ≥24.6 CD4 mm<sup>3</sup>/ml, sensitivity 73.68%, specificity 73.68%. <b></b></span><b><b><span style="font-family:Verdana;">Conclusions:</span></b><span style="font-family:Verdana;"></span></b> The ESAT-6 detection assay using NIPAm was effective, with higher rates in patients with ≤200 CD4 cells/mm<sup>3</sup>, the test being more sensitive than smear and culture, but less specific.</span></span></span>展开更多
Purpose: There is a need for a continued effort to develop rapid immunodiagnostic assays for tuberculosis (TB) infection with greater sensitivity and specificity that can be used in the field and in the laboratory and...Purpose: There is a need for a continued effort to develop rapid immunodiagnostic assays for tuberculosis (TB) infection with greater sensitivity and specificity that can be used in the field and in the laboratory and that can be formatted for use with multiple species. This would help to obtain definitive early diagnosis of TB. The present study was developed to determine the role of using early secreted antigenic target-6 (ESAT-6) in immunodiagnosis of Mycobacterium tuberculosis. Methods: Serum samples were obtained from TB infected patients and normal healthy controls. Two rapid immunodiagnostic assays (Enzyme-linked immunosorbent assay (ELISA) and Immunoblotting) were performed. Results: The sensitivity of immunoblotting assay was 100%;however, ESAT-6 antigen was not able to discriminate between patients and normal controls. Application of direct ELISA using ESAT-6 antigen yielded 97.6% sensitivity and 75% specificity for the diagnosis of TB infection. Conclusion: In conclusion, the detection of antibodies against ESAT-6 antigen in the sera of TB patients by direct ELISA could be used as a preliminary assay for diagnosis of human M. tuberculosis infection. A combination of the ELISA with either radiological or microscopic examination is required to overcome the low specificity of the assay for negative results.展开更多
Background Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guerin （BCG） is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing ne...Background Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guerin （BCG） is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing new tuberculosis vaccines becomes an urgent task. In this study, we evaluated in BALB/c mice the humoral and cellular immune responses of recombinant BCG expressing the antigen ESAT-6 from Mycobacterium tuberculosis.Methods Escherichia coli-BCG shuttle plasmid named pDE22-esat-6 was constructed by inserting the BamHI/EcoRI digested esat-6 gene PCR product into the similarly digested parental plasmid pDE22. BCG cells were transformed with pDE22-esat-6, which was named recombinant BCG （rBCG）. BALB/c mice were immunized subcutaneously on the back with 100 μl normal saline containing 106 CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses. Results There was no any significant differences in the growth characteristics between the conventional BCG and rBCG. In immunized mice, the IgG antibody titres of rBCG group were as high as 1：8000, which was significantly higher than that in BCG group （1：1400, P〈0.05）. The elicited IFN-γ, level of rBCG group was （1993 ± 106） pg/ml, which was also significantly higher than that in BCG group （（1463 ± 105） pg/ml, P〈0.05）. The splenocyte proliferation index of rBCG group reached 4.34 ± 0.31, which was higher than that of BCG group （3.79 ±0.24, P〈0.05）. Conclusion rBCG secreted expressing antigen ESAT-6 stimulated stronger humoral and cellular immune responses than BCG did, and, therefore may be the better vaccine against mycobacterium tuberculosis.展开更多
The flagellin component FliC of Salmonella typhimurium is capable of activating the innate immune system via specific interactions with TLR5 and can also act as a carrier of foreign antigen to elicit antigen-specific ...The flagellin component FliC of Salmonella typhimurium is capable of activating the innate immune system via specific interactions with TLR5 and can also act as a carrier of foreign antigen to elicit antigen-specific immune responses.Thus,we constructed an attenuated Salmonella strain SL5928(fliC/esat)expressing chimeric flagellin that contained the ESAT-6 antigen coding sequence of Mycobacterium tuberculosis inserted into the highly variable region of the Salmonella flagellin coding gene fliCi.The chimeric flagellin functioned normally,as demonstrated using a flagella swarming assay and electron microscopy.To analyze the effects of chimeric flagellin,the cell-mediated immune response and cytotoxic T lymphocyte(CTL)effects specific for ESAT-6 antigen were tested after intranasal immunization of mice with flagellated Salmonella SL5928(fliC/esat).The results showed that SL5928(fliC/esat)intranasal immunization can strongly elicit an ESAT-6-specific T helper(Th)1-type immune response in mucosal lymphoid tissues,such as nasopharynx-associated lymph nodes,lung and Peyer’s patches,and a Th1/Th2 response was elicited in spleen and mesenteric lymph nodes.Furthermore,intranasal immunization of SL5928(fliC/esat)produced efficient CTL effects,as demonstrated using a 5-and 6-carboxyfluorescein diacetate succinimidyl ester(CFSE)assay.Thus,our study revealed that Salmonella flagellin acts as a carrier for foreign antigen and triggers strong Th1 and CTL responses during intranasal immunization.Chimeric flagellin is potentially an effective strategy for the development of novel vaccines against tuberculosis in humans and animals.展开更多
目的检测不同人群血浆标本中白细胞介素-6(IL-6)的表达情况,探讨其在结核病诊断中的临床意义。方法以蛋白芯片筛选结核诊断标志物时发现IL-6在结核患者中高表达,扩大样本采用酶联免疫吸附试验(ELISA)法检测39例实验室确诊结核病组(patie...目的检测不同人群血浆标本中白细胞介素-6(IL-6)的表达情况,探讨其在结核病诊断中的临床意义。方法以蛋白芯片筛选结核诊断标志物时发现IL-6在结核患者中高表达,扩大样本采用酶联免疫吸附试验(ELISA)法检测39例实验室确诊结核病组(patients with tuberculosis,TB)、31例结核潜伏感染组(latent tuberculosis infection,LTBI)及31例健康对照组(healthy control,HC)3组人群血浆中IL-6水平的差异,以评价IL-6对结核病的效能。结果蛋白芯片检测发现IL-6在TB组、LTBI组和HCTB组中表达分别为(550.39±479.82)pg/mL、(16.67±12.95)pg/mL、(126.89±84.75)pg/mL,TB组明显高于LTBI组及HC组,差异有统计学意义(P<0.01)。以ELISA,TB组、LTBI组和HCTB组中表达分别为(8359.85±7487.13)pg/mL、(1102.54±2079.32)pg/mL、(1873.60±4320.03)pg/mL,TB组明显高于LTBI组及HC组,差异有统计学意义(P<0.01)。分辨TB和HC的效能曲线下面积(Area Under Curve,AUC)为0.8577,灵敏度为79.49%,特异度为80.65%;分辨TB和LTBI的效能AUC为0.8834,灵敏度为92.31%,特异度为70.97%。结核患者血浆中IL-6的表达水平显著高于健康对照和结核潜伏感染人群(P<0.01)。结论结核患者血浆中IL-6的表达显著高于健康对照和潜伏感染者,IL-6可能作为用于区分活动性结核与潜伏结核感染人群的生物标识,用于早期诊断。展开更多
文摘<b><span style="font-family:Verdana;">Background:</span></b><span style="font-family:Verdana;"> In patients with HIV, conventional tests are of low sensitivity;therefore, a new diagnostic test with hydrogel nanoparticles with reactive blue dye is proposed, which allows the capture, conservation and concentration of ESAT-6 in urine. NIPAs are copolymers that capture low molecular weight proteins and protect against enzymatic degradation. Using an immunoassay, it is possible to detect and quantify ESAT-6 present in urine as a possible marker of active TB. <b></b></span><b><b><span style="font-family:Verdana;">Design/Methods:</span></b><span style="font-family:Verdana;"></span></b> Study in Lima, Peru, HIV+ participants, ≥18 years with and without tuberculosis (TB). Smear microscopy, culture in solid medium and urine immunoassay were performed. The reference was the diagnosis of TB by radiological or clinical microbiological criteria (indication for TB treatment). There were 2 preanalytical processes: untreated and treated urine (centrifuged, heated), then incubation with NIPAm. After washing, elution, sonication, heat and centrifugation, the final eluate was obtained. This was placed on nitrocellulose membranes, which by means of fixation and incubation processes with anti-ESAT-6 and anti-IgG antibodies, revelation with C-DiGit<span style="white-space:nowrap;">®</span> Blot Scanner and FluorChem R FR0001. Calibration curves were included in the membranes, density was measured using Image J software. ROC curves, sensitivity and specificity were obtained. <b><b><span style="font-family:Verdana;">Results:</span></b><span style="font-family:Verdana;"></span></b> The result by groups was HIV+ patient: ROC: 0.75<span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">,</span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">c</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">ut-off point ≥24.06 ng/ml, sensitivity 76.32%, specificity 68.89%, patients ≤200 cells CD4 mm<sup>3</sup>/ml, ROC: 0.78, cutoff point ≥26.20 ng/ml, sensitivity 75.86%, specificity 71.88%, patients</span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span><span><span style="font-family:;" "=""><span style="font-family:Verdana;">>200 CD4 mm</span><sup><span style="font-family:Verdana;">3</span></sup><span style="font-family:Verdana;"> cells/ml, ROC</span></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">:</span></span></span><span><span><span style="font-family:;" "=""><span style="font-family:Verdana;"> 0.73, cutoff point ≥24.6 CD4 mm<sup>3</sup>/ml, sensitivity 73.68%, specificity 73.68%. <b></b></span><b><b><span style="font-family:Verdana;">Conclusions:</span></b><span style="font-family:Verdana;"></span></b> The ESAT-6 detection assay using NIPAm was effective, with higher rates in patients with ≤200 CD4 cells/mm<sup>3</sup>, the test being more sensitive than smear and culture, but less specific.</span></span></span>
文摘Purpose: There is a need for a continued effort to develop rapid immunodiagnostic assays for tuberculosis (TB) infection with greater sensitivity and specificity that can be used in the field and in the laboratory and that can be formatted for use with multiple species. This would help to obtain definitive early diagnosis of TB. The present study was developed to determine the role of using early secreted antigenic target-6 (ESAT-6) in immunodiagnosis of Mycobacterium tuberculosis. Methods: Serum samples were obtained from TB infected patients and normal healthy controls. Two rapid immunodiagnostic assays (Enzyme-linked immunosorbent assay (ELISA) and Immunoblotting) were performed. Results: The sensitivity of immunoblotting assay was 100%;however, ESAT-6 antigen was not able to discriminate between patients and normal controls. Application of direct ELISA using ESAT-6 antigen yielded 97.6% sensitivity and 75% specificity for the diagnosis of TB infection. Conclusion: In conclusion, the detection of antibodies against ESAT-6 antigen in the sera of TB patients by direct ELISA could be used as a preliminary assay for diagnosis of human M. tuberculosis infection. A combination of the ELISA with either radiological or microscopic examination is required to overcome the low specificity of the assay for negative results.
基金This work was supported by the grants from the National Natural Science Foundation of China (No. 30400381)the "863"Program(No. 2001 AA215201).
文摘Background Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guerin （BCG） is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing new tuberculosis vaccines becomes an urgent task. In this study, we evaluated in BALB/c mice the humoral and cellular immune responses of recombinant BCG expressing the antigen ESAT-6 from Mycobacterium tuberculosis.Methods Escherichia coli-BCG shuttle plasmid named pDE22-esat-6 was constructed by inserting the BamHI/EcoRI digested esat-6 gene PCR product into the similarly digested parental plasmid pDE22. BCG cells were transformed with pDE22-esat-6, which was named recombinant BCG （rBCG）. BALB/c mice were immunized subcutaneously on the back with 100 μl normal saline containing 106 CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses. Results There was no any significant differences in the growth characteristics between the conventional BCG and rBCG. In immunized mice, the IgG antibody titres of rBCG group were as high as 1：8000, which was significantly higher than that in BCG group （1：1400, P〈0.05）. The elicited IFN-γ, level of rBCG group was （1993 ± 106） pg/ml, which was also significantly higher than that in BCG group （（1463 ± 105） pg/ml, P〈0.05）. The splenocyte proliferation index of rBCG group reached 4.34 ± 0.31, which was higher than that of BCG group （3.79 ±0.24, P〈0.05）. Conclusion rBCG secreted expressing antigen ESAT-6 stimulated stronger humoral and cellular immune responses than BCG did, and, therefore may be the better vaccine against mycobacterium tuberculosis.
基金supported by grants from the Major State Basic Research Development Program of China(973 Program)the National S T Major Project(no.2008 ZX10003-010)+2 种基金the National Department Public Benefit Research Foundation(200903027)the Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT)the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
文摘The flagellin component FliC of Salmonella typhimurium is capable of activating the innate immune system via specific interactions with TLR5 and can also act as a carrier of foreign antigen to elicit antigen-specific immune responses.Thus,we constructed an attenuated Salmonella strain SL5928(fliC/esat)expressing chimeric flagellin that contained the ESAT-6 antigen coding sequence of Mycobacterium tuberculosis inserted into the highly variable region of the Salmonella flagellin coding gene fliCi.The chimeric flagellin functioned normally,as demonstrated using a flagella swarming assay and electron microscopy.To analyze the effects of chimeric flagellin,the cell-mediated immune response and cytotoxic T lymphocyte(CTL)effects specific for ESAT-6 antigen were tested after intranasal immunization of mice with flagellated Salmonella SL5928(fliC/esat).The results showed that SL5928(fliC/esat)intranasal immunization can strongly elicit an ESAT-6-specific T helper(Th)1-type immune response in mucosal lymphoid tissues,such as nasopharynx-associated lymph nodes,lung and Peyer’s patches,and a Th1/Th2 response was elicited in spleen and mesenteric lymph nodes.Furthermore,intranasal immunization of SL5928(fliC/esat)produced efficient CTL effects,as demonstrated using a 5-and 6-carboxyfluorescein diacetate succinimidyl ester(CFSE)assay.Thus,our study revealed that Salmonella flagellin acts as a carrier for foreign antigen and triggers strong Th1 and CTL responses during intranasal immunization.Chimeric flagellin is potentially an effective strategy for the development of novel vaccines against tuberculosis in humans and animals.
文摘目的检测不同人群血浆标本中白细胞介素-6(IL-6)的表达情况,探讨其在结核病诊断中的临床意义。方法以蛋白芯片筛选结核诊断标志物时发现IL-6在结核患者中高表达,扩大样本采用酶联免疫吸附试验(ELISA)法检测39例实验室确诊结核病组(patients with tuberculosis,TB)、31例结核潜伏感染组(latent tuberculosis infection,LTBI)及31例健康对照组(healthy control,HC)3组人群血浆中IL-6水平的差异,以评价IL-6对结核病的效能。结果蛋白芯片检测发现IL-6在TB组、LTBI组和HCTB组中表达分别为(550.39±479.82)pg/mL、(16.67±12.95)pg/mL、(126.89±84.75)pg/mL,TB组明显高于LTBI组及HC组,差异有统计学意义(P<0.01)。以ELISA,TB组、LTBI组和HCTB组中表达分别为(8359.85±7487.13)pg/mL、(1102.54±2079.32)pg/mL、(1873.60±4320.03)pg/mL,TB组明显高于LTBI组及HC组,差异有统计学意义(P<0.01)。分辨TB和HC的效能曲线下面积(Area Under Curve,AUC)为0.8577,灵敏度为79.49%,特异度为80.65%;分辨TB和LTBI的效能AUC为0.8834,灵敏度为92.31%,特异度为70.97%。结核患者血浆中IL-6的表达水平显著高于健康对照和结核潜伏感染人群(P<0.01)。结论结核患者血浆中IL-6的表达显著高于健康对照和潜伏感染者,IL-6可能作为用于区分活动性结核与潜伏结核感染人群的生物标识,用于早期诊断。
