实时荧光定量PCR对结核病潜伏感染快速诊断的研究

A study of a technique to rapidly identify a latent Mycobacterium tuberculosis infection

目的探讨7种细胞因子的mRNA表达水平作为诊断结核分枝杆菌潜伏感染生物标志物的可能性。方法健康对照64人,活动性肺结核50人,结核分枝杆菌潜伏感染60人,采用实时荧光定量PCR(qRT-PCR)检测结核特异性抗原刺激的外周血中TNF-α、IFN-γ、IL-2、IL-10、IFI35、CXCL10及Foxp3的mRNA表达水平。结果 7种因子相对表达量在活动性肺结核组分别为2.33±0.09、4.94±0.45、150±17.82、2.02±0.55、9.53±5.96、5.56±0.97、1.24±0.17,潜伏感染组分别为3.2±0.61、10.04±1.89、202±17.6、21.89±10.25、29.17±11.19、41.35±11.46、2.14±0.67,健康组分别为1.2±0.08、2.92±0.03、53±17.82、1.48±0.17、1.28±0.77、1.63±0.25、1.03±0.67。潜伏感染组与健康组相比7种细胞因子的mRNA表达水平,差异均有统计学意义(P<0.01),相应细胞因子检测的敏感性、特异性分别为95.1%、82.5%,92.5%、88.7%,91.9%、90.1%,88.2%、51.5%,74.2%、61.2%,81.8%、73.4%和80.4%、51.3%;潜伏感染组和活动性结核组比较除IFI35因子外,其余6种细胞因子的mRNA表达水平差异均有统计学意义(P<0.05),相应细胞因子检测的特异性、敏感性分别为90.6%、80%,88.6%、92.7%,91.9%、86.2%,57.6%、60%,50%、58%,76%、72.2%和66.7%、67.1%;健康对照组和活动性结核组相比较,7种细胞因子的mRNA表达水平差异均有统计学意义(P<0.01),相应细胞因子检测的敏感性、特异性分别为92.3%、90%,88.6%、86.7%,94.4%、86.7%,89.7%、65.8%,82.4%、63.3%,88.2%、70%和92.9%、72.1%。结核病密切接触者潜伏感染率为48.97%,健康组潜伏感染率为36%。结论结核病患者血清TNF-α、IFN-γ、IL-2mRNA水平升高,以潜伏感染者升高更显著,因此可作为诊断结核分枝杆菌潜伏感染的生物标志物。

Objective To investigate the potential for levels of mRNA expression of 6cytokines（TNF-α,IFN-γ,IL-2,IL-10,IFI35,and CXCL10）and 1transcription factor（Foxp3）to serve as biomarkers for diagnosis of a latent TB infection. Methods Subjects were 64 healthy controls,50 patients with active tuberculosis（TB）,and 60 patients with a latent Mycobacterium tuberculosis infection.After stimulation with M.tuberculosis-specific antigens,peripheral blood from the 3groups of subjects was tested for levels of expression of TNF-α,IFN-γ,IL-2,IL-10,IFI35,CXCL10,and Foxp3 mRNA using real-time quantitative PCR（qRT-PCR）. Results In patients with active TB,the level of expression of TNF-αmRNA was 2.33±0.09,the level of expression of IFN-γmRNA was 4.94±0.45,the level of expression of IL-2mRNA was 150±17.82,the level of expression of IL-10 mRNA was 2.02±0.55,the level of expression of IFI35 mRNA was 9.53±5.96,the level of expression of CXCL10 mRNA was 5.56±0.97,and the level of expression of Foxp3 mRNA was 1.24±0.17.In patients with a latent M.tuberculosisinfection,the level of expression of TNF-αmRNA was3.2±0.61,the level of expression of IFN-γmRNA was 10.04±1.89,the level of expression of IL-2mRNA was 202±17.6,the level of expression of IL-10 mRNA was 21.89±10.25,the level of expression of IFI35 mRNA was 29.17±11.19,the level of expression of CXCL10 mRNA was 41.35±11.46,and the level of expression of Foxp3 mRNA was2.14±0.67.In healthy controls,the level of expression of TNF-αmRNA was 1.2±0.08,the level of expression of IFN-γmRNA was 2.92±0.03,the level of expression of IL-2mRNA was 53±17.82,the level of expression of IL-10 mRNA was 1.48±0.17,the level of expression of IFI35 mRNA was 1.28±0.77,the level of expression of CXCL10 mRNA was1.63±0.25,and the level of expression of Foxp3 mRNA was 1.03±0.67.The levels of mRNA expression of the 6cytokines and 1transcription factor differed significantly（P〈0.01）in patients with a latent infection and healthy controls.TNF-αhad a sensitivity of 95.1% and a specificity of 82.5%,IFN-γhad a sensitivity of 92.5% and a specificity of88.7%,IL-2had a sensitivity of 91.9% and a specificity of 90.1%,IL-10 had a sensitivity of 88.2% and a specificity of51.5%,IFI35 had a sensitivity of 74.2%and a specificity of 61.2%,CXCL10 had a sensitivity of 81.8%and a specificity of 73.4%,and Foxp3 had a sensitivity of 80.4% and a specificity of 51.3%.Levels of mRNA expression of 5cytokines（not including IFI35）and 1transcription factor differed significantly（P〈0.05）in patients with a latent infection and patients with active TB.TNF-αhad a sensitivity of 90.6% and a specificity of 80%,IFN-γhad a sensitivity of88.6% and a specificity of 92.7%,IL-2had a sensitivity of 91.9% and a specificity of 86.2%,IL-10 had a sensitivity of57.6% and a specificity of 60%,IFI35 had a sensitivity of 50% and a specificity of 58%,CXCL10 had a sensitivity of76% and a specificity of 72.2%,and Foxp3 had a sensitivity of 66.7% and a specificity of 67.1%.Levels of mRNA expression of the 6cytokines and 1transcription factor differed significantly（P〈0.01）in patients with active TB and healthy controls.TNF-αhad a sensitivity of 92.3%and a specificity of 90%,IFN-γhad a sensitivity of 88.6%and a specificity of 86.7%,IL-2had a sensitivity of 94.4% and a specificity of 86.7%,IL-10 had a sensitivity of 89.7% and a specificity of 65.8%,IFI35 had a sensitivity of 82.4% and a specificity of 63.3%,CXCL10 had a sensitivity of 88.2%and a specificity of 70%,and Foxp3 had a sensitivity of 92.9% and a specificity of 72.1%.The rate of a latent infection was 48.97%in individuals in close contact with M.tuberculosis and 36%in health group. Conclusion Levels of TNF-α,IFN-γ,and IL-2mRNA expression were higher in patients with TB and were markedly higher in patients with a latent infection.Therefore,the levels of these 3cytokines can be used as biomarkers for diagnosis of a latent M.tuberculosisinfection.