一.干扰素释放试验对于结核性脑膜炎的诊断价值以及诊断模型的建立 二.结核性脑膜炎差异miRNA的初步筛选
发布时间：2018-09-18 17:42
【摘要】：TB目前仍然是严重威胁人类生命的最主要感染性疾病，中国2010年全国结核病流行病学调查显示肺结核病例约500万，每年新发病例达100万。结核性脑膜炎（tuberculous meningitis，TBM）的发病率虽然仅占结核病的1%-2%，但是其死亡率和致残率却高于40%，，高死亡率主要是缺乏敏感特异的诊断手段所致。因此，寻找一种新的敏感特异的诊断方法，提高TBM的诊断水平，是降低TBM死亡率和致残率的关键。 γ-干扰素释放试验（interferon gamma releasing assay，IGRAs）根据特异性抗原刺激后机体效应T淋巴细胞产生的γ-干扰素来判断诊断结核感染的免疫学方法，由于不能区分结核潜伏感染（latent tuberculosis infection，LTBI）和活动性结核病（active tuberculosis，ATB），而致其在TB高负担国家诊断价值受限。结核分枝杆菌（Mycobacterium Tuberculosis，M.TB）感染部位具有隔室化的特点，即抗原特异性T淋巴细胞被募集到局部感染部位，感染部位如脑脊液（cerebrospinal fluid，CSF）的T-SPOT.TB有可能获得比外周血（peripheral blood，PB) T-SPOT.TB更高的诊断准确性。目前我国尚没有CSF T-SPOT.TB检测诊断TBM的相关数据。 Thwaites GE等2002年通过五种临床特征建立了预测TBM的越南模型。模型包含五项内容：年龄、病程、血常规白细胞计数、CSF白细胞计数和CSF中性粒细胞比例。越南模型主要是鉴别TBM和细菌性脑膜炎，而在我国细菌性脑膜炎发病率相对较低，因此这一模型并不一定符合我国脑膜炎的发病现状，还需要建立适合我国的一种新的诊断模型。由于PB T-SPOT.TB和CSFT-SPOT.TB可能具有较高的诊断准确性，将这两项指标以及其他临床检测指标联合，建立诊断模型，有可能获得较好的诊断准确性。 本课题最终纳入TBM31例和non-TBM40例进行PB T-SPOT.TB和CSFT-SPOT.TB，两种检测的敏感性分别为93.6%（29/31,95%CI：79.3%-98.2%）和53.3%（16/30，95%CI：36.1%-70.0%），特异性分别为75.0%（30/40，95%CI：59.8%-85.8%）和94.8%（37/39，95%CI：83.1%-98.6%），诊断准确性分别为83.7%（59/71,72.7%-90.1%）和76.8%（53/69，95%CI：65.6%-85.2%）。PB T-SPOT诊断的敏感性高于CSF T-SPOT.TB，而CSF T-SPOT.TB的特异性高于PBT-SPOT.TB。将这两项指标和TBM、non-TBM之间存在差异的CSF蛋白、CSF腺苷脱氨酶和发病时间等共五项指标输入支持向量机（SVM），优选出的最具诊断意义的是PB T-SPOT.TB和CSF T-SPOT.TB，用这两项指标确立决策树、建立联合诊断模型，得到联合诊断模型的敏感性和特异性分别为90.0%（27/30，95%CI：27/30）和94.9%（37/39，95%CI：83.1%-98.6%），其诊断准确性为92.8%（64/69，95%CI：84.8%-96.9%），高于PB T-SPOT.TB的和CSF T-SPOT.TB。而从本课题纳入的病例分析，越南模型验证的特异性仅为5%。 本研究表明PB T-SPOT.TB由于敏感性较高，有可能作为排除TBM诊断的一种方法，CSF T-SPOT.TB由于特异性较高，有可能作为目前纳入TBM诊断的一种方法。越南模型并不适用于我国TBM的诊断和鉴别诊断，而根据PBT-SPOT.TB和CSF T-SPOT.TB建立的联合诊断模型对于结核性脑膜炎的诊断具有较高的诊断价值，但是无论单一的PB T-SPOT.TB和CSF T-SPOT.TB检测，或及联合诊断模型的意义，都需要进一步进行大样本前瞻性实验加以验证。 miRNA是近年来生命科学研究的热点，某些miRNA或可作为肿瘤、自身免疫性疾病诊断的生物标识。然而，miRNA作为生物标识在细菌感染性疾病尤其是TB中的研究相对较少。由于这种小分子在细胞的生长、分化、凋亡、代谢以及病毒感染、肿瘤的发生等生理病理过程中具有重要作用，因此推测其在TBM发病的免疫学机制、疾病的进展等方面也可能具有一定的意义，有可能做为TBM诊断的生物标识。诊断手段的缺乏是导致TBM的死亡率多年来高居不下的首要原因，如果找到特异表达的miRNA，则可以提高TBM的诊断水平。 本研究入选TBM组8例，non-TBM（病毒性脑膜炎）组8例和健康对照6例，应用Agilent芯片筛选得到11种差异miRNA，具体为hsa-miR-126-3p/5p，hsa-miR-130a-3p， hsa-miR-151a-3p/5p， hsa-miR-199a-3p， hsa-miR-221-3p，hsa-miR-4291，hsa-miR-584-5p，hsa-miR-6127，hsa-miR-744-5p。通过靶基因预测和GO分析发现上述差异miRNA的靶基因的功能主要是参与转录过程、DNA与RNA的代谢过程等；Pathway通路分析显示miRNA的靶基因主要是参与肿瘤通路、MAPK信号通路、Wnt通路等。今后的工作中将进一步筛选病例扩大样本量进行RT-PCR验证，以确定差异miRNA是否具有诊断TBM的意义，同时要进一步的研究以证实这些差异miRNA是否与炎症反应以及TB、TBM发病的免疫机制相关。
[Abstract]:Tuberculous meningitis (TBM) is still the most serious infectious disease that threatens human life. The epidemiological survey of tuberculosis in China in 2010 showed that about 5 million cases of tuberculosis and 1 million new cases are reported every year. Although the incidence of tuberculous meningitis (TBM) is only 1% - 2% of tuberculosis, its mortality and disability rate are high. At 40%, the high mortality rate is mainly due to the lack of sensitive and specific diagnostic methods. Therefore, finding a new sensitive and specific diagnostic method to improve the diagnostic level of TBM is the key to reduce the mortality and disability rate of TBM.
Interferon gamma release assay (IGRAs) is an immunological method for the diagnosis of tuberculosis infection based on interferon-gamma produced by effector T lymphocytes stimulated by specific antigens. It can not distinguish latent tuberculosis infection (LTBI) from active tuberculosis (active tuberculosis). Mycobacterium tuberculosis (M. TB) infection site has the characteristics of compartmentalization, that is, antigen-specific T lymphocytes are recruited to locally infected sites, infection sites such as cerebrospinal fluid (CSF) T-SPOT. TB may obtain more than peripheral blood (periph). The diagnostic accuracy of PB T-SPOT.TB is higher than that of PB T-SPOT.TB.
Thwaites GE et al. established a Vietnamese model for predicting TBM through five clinical features in 2002. The model consists of five items: age, course of disease, blood routine white blood cell count, CSF white blood cell count and CSF neutrophil ratio. Because PB T-SPOT.TB and CSFT-SPOT.TB may have higher diagnostic accuracy, it is possible to combine these two indicators with other clinical detection indicators to establish a diagnostic model, which may obtain better diagnostic accuracy. Sex.
The sensitivity of PB T-SPOT.TB and CSFT-SPOT.TB were 93.6% (29/31, 95% CI: 79.3% - 98.2%) and 53.3% (16/30, 95% CI: 36.1% - 70.0%) respectively, and the specificity was 75.0% (30/40, 95% CI: 59.8% - 85.8%) and 94.8% (37/39, 95% CI: 83.1% - 98.6%) respectively. The diagnostic accuracy was 57.7% (59/39, 95% CI: 83.1% - 98.6%). The sensitivity of PBT-SPOT was higher than that of CSF T-SPOT.TB, and the specificity of CSF T-SPOT.TB was higher than that of PBT-SPOT. PB T-SPOT.TB and CSF T-SPOT.TB were used to establish decision tree and joint diagnosis model. The sensitivity and specificity of the combined diagnosis model were 90.0% (27/30, 95% CI: 27/30) and 94.9% (37/39, 95% CI: 83.1% - 98.6%) respectively. The diagnostic accuracy was 92.8% (64/69, 95% CI: 84.8% - 96.9%) higher than that of PB T-SPOT. The specificity of B model and CSF T-SPOT.TB. was only 5%. for the case analysis.
This study indicates that PBT-SPOT.TB may be used as a method to exclude TBM because of its high sensitivity and specificity. CSF T-SPOT.TB may be used as a method to diagnose TBM at present. Vietnam model is not suitable for the diagnosis and differential diagnosis of TBM in China, but the combined diagnosis based on PBT-SPOT.TB and CSF T-SPOT.TB. The severed model has a high diagnostic value for tuberculous meningitis, but the significance of single PB T-SPOT.TB and CSF T-SPOT.TB test, or combined diagnosis model, needs to be further validated by large-sample prospective experiments.
MicroRNAs are the hotspot of life science research in recent years. Some microRNAs may be used as biomarkers for the diagnosis of tumors and autoimmune diseases. However, there are relatively few studies on microRNAs as biomarkers in bacterial infectious diseases, especially in TB. It may be a biomarker for the diagnosis of TBM. The lack of diagnostic tools is the primary cause of the high mortality rate of TBM over the years. If the specific expression of microRNAs can be found, it can be used as a biomarker for the diagnosis of TBM. NA can improve the diagnostic level of TBM.
In this study, 8 TBM patients, 8 non-TBM (viral meningitis) patients, 8 non-TBM (viral meningitis) patients and 6 healthy controls were enrolled, 11 different microRNAs were screened by Agilent chip, including hsa-microRNA-126-3p/5p, hsa-microRNA-130a-3p, hsa-microRNA-151a-3p/5p, hsa-microRNA-199a-199a-3p, hsa-microRNA-199a-199a-199a-3p, hsa-microRNA-4291, hsa-microarray-microRNA-584-584-5p, hsa-5p, hsa-microRNA-614-5p, hsa-61a-61a-61a-61a-61a P. Pre-gene by Target The results of GO analysis showed that the target genes of these differentially expressed microRNAs were mainly involved in transcription, DNA and RNA metabolism, and Pathway pathway analysis showed that the target genes of microRNAs were mainly involved in tumor pathway, MAPK signaling pathway and Wnt pathway. To determine whether differentially expressed microRNAs have diagnostic significance for TBM, further studies are needed to confirm whether these differentially expressed microRNAs are associated with inflammation and the immune mechanism of TB and TBM.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R529.3
本文编号：2248646
[Abstract]:Tuberculous meningitis (TBM) is still the most serious infectious disease that threatens human life. The epidemiological survey of tuberculosis in China in 2010 showed that about 5 million cases of tuberculosis and 1 million new cases are reported every year. Although the incidence of tuberculous meningitis (TBM) is only 1% - 2% of tuberculosis, its mortality and disability rate are high. At 40%, the high mortality rate is mainly due to the lack of sensitive and specific diagnostic methods. Therefore, finding a new sensitive and specific diagnostic method to improve the diagnostic level of TBM is the key to reduce the mortality and disability rate of TBM.
Interferon gamma release assay (IGRAs) is an immunological method for the diagnosis of tuberculosis infection based on interferon-gamma produced by effector T lymphocytes stimulated by specific antigens. It can not distinguish latent tuberculosis infection (LTBI) from active tuberculosis (active tuberculosis). Mycobacterium tuberculosis (M. TB) infection site has the characteristics of compartmentalization, that is, antigen-specific T lymphocytes are recruited to locally infected sites, infection sites such as cerebrospinal fluid (CSF) T-SPOT. TB may obtain more than peripheral blood (periph). The diagnostic accuracy of PB T-SPOT.TB is higher than that of PB T-SPOT.TB.
Thwaites GE et al. established a Vietnamese model for predicting TBM through five clinical features in 2002. The model consists of five items: age, course of disease, blood routine white blood cell count, CSF white blood cell count and CSF neutrophil ratio. Because PB T-SPOT.TB and CSFT-SPOT.TB may have higher diagnostic accuracy, it is possible to combine these two indicators with other clinical detection indicators to establish a diagnostic model, which may obtain better diagnostic accuracy. Sex.
The sensitivity of PB T-SPOT.TB and CSFT-SPOT.TB were 93.6% (29/31, 95% CI: 79.3% - 98.2%) and 53.3% (16/30, 95% CI: 36.1% - 70.0%) respectively, and the specificity was 75.0% (30/40, 95% CI: 59.8% - 85.8%) and 94.8% (37/39, 95% CI: 83.1% - 98.6%) respectively. The diagnostic accuracy was 57.7% (59/39, 95% CI: 83.1% - 98.6%). The sensitivity of PBT-SPOT was higher than that of CSF T-SPOT.TB, and the specificity of CSF T-SPOT.TB was higher than that of PBT-SPOT. PB T-SPOT.TB and CSF T-SPOT.TB were used to establish decision tree and joint diagnosis model. The sensitivity and specificity of the combined diagnosis model were 90.0% (27/30, 95% CI: 27/30) and 94.9% (37/39, 95% CI: 83.1% - 98.6%) respectively. The diagnostic accuracy was 92.8% (64/69, 95% CI: 84.8% - 96.9%) higher than that of PB T-SPOT. The specificity of B model and CSF T-SPOT.TB. was only 5%. for the case analysis.
This study indicates that PBT-SPOT.TB may be used as a method to exclude TBM because of its high sensitivity and specificity. CSF T-SPOT.TB may be used as a method to diagnose TBM at present. Vietnam model is not suitable for the diagnosis and differential diagnosis of TBM in China, but the combined diagnosis based on PBT-SPOT.TB and CSF T-SPOT.TB. The severed model has a high diagnostic value for tuberculous meningitis, but the significance of single PB T-SPOT.TB and CSF T-SPOT.TB test, or combined diagnosis model, needs to be further validated by large-sample prospective experiments.
MicroRNAs are the hotspot of life science research in recent years. Some microRNAs may be used as biomarkers for the diagnosis of tumors and autoimmune diseases. However, there are relatively few studies on microRNAs as biomarkers in bacterial infectious diseases, especially in TB. It may be a biomarker for the diagnosis of TBM. The lack of diagnostic tools is the primary cause of the high mortality rate of TBM over the years. If the specific expression of microRNAs can be found, it can be used as a biomarker for the diagnosis of TBM. NA can improve the diagnostic level of TBM.
In this study, 8 TBM patients, 8 non-TBM (viral meningitis) patients, 8 non-TBM (viral meningitis) patients and 6 healthy controls were enrolled, 11 different microRNAs were screened by Agilent chip, including hsa-microRNA-126-3p/5p, hsa-microRNA-130a-3p, hsa-microRNA-151a-3p/5p, hsa-microRNA-199a-199a-3p, hsa-microRNA-199a-199a-199a-3p, hsa-microRNA-4291, hsa-microarray-microRNA-584-584-5p, hsa-5p, hsa-microRNA-614-5p, hsa-61a-61a-61a-61a-61a P. Pre-gene by Target The results of GO analysis showed that the target genes of these differentially expressed microRNAs were mainly involved in transcription, DNA and RNA metabolism, and Pathway pathway analysis showed that the target genes of microRNAs were mainly involved in tumor pathway, MAPK signaling pathway and Wnt pathway. To determine whether differentially expressed microRNAs have diagnostic significance for TBM, further studies are needed to confirm whether these differentially expressed microRNAs are associated with inflammation and the immune mechanism of TB and TBM.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R529.3
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