医学ppt课件大全结核病实验室诊断技术及其进展

结核病实验室诊断技术及其进展 中国疾病预防控制中心结核病防治临床中心 国家结核病参比实验室 赵雁林 结核病的诊断 实验室诊断需要关注的问题 WHO推荐的肺结核的诊断标准 肺结核的实验室诊断 结核病的实验室诊断新技术 内 容 1. 结构基因组学向功能基因组学转变 2. 基因组学向蛋白组学转变 3. 以作图为基础的基因鉴定向以序列为基础的 基因鉴定转变 4. 单基因病研究向多基因病研究转变 生物医学研究模式发生变化趋势 生物医学研究模式发生变化趋势（续 ） 5. 对疾病的特异性 DNA诊断向疾病易感性鉴定转变 6. 分析单个基因作用向研究基因作用的调控机制转变 7. 研究疾病的病因向研究疾病的病理发生机制转变 8. 研究单一种属向研究多种种属转变。 M. tuberculosis H37Rv Cole ST et al. Nature 1998; 393: 537. 441 .1万碱基对 3924 预测基因 （ 3995 predicted protein-coding genes) 65.6 % GC 含量 重复 DNA Repetitive DNA in the Mycobacterium tuberculosis Complex 分类： 1.串联重复序列 Tandem Repeats(TRs） 卫星 DNA、 小卫星 DNA、 微卫星 DNA VNTR 2. 散在重复序列 Interpersed Repeats GC富含重复序列和主要的串联重复序列 Polymorphic GC-Rich Repetitive Sequence 57:275-99 结核病变在体内的演变和播散 传染途径 结核病的传染系由细小 飞沫而 发生 饮用来自病牛的牛奶而罹患肠结核 在没有 HIV感染的人群中，结核杆菌感染者在其一生中 大约有 10%的人会发病 In the absence of HIV, about 10% of people infected with TB will develop TB disease. 在 TB/HIV双重感染者中约 50%的感染者会成为结核病患者 In people co-infected with HIV and TB, about 50 % may develop TB disease TB 加速和恶化 HIV/AIDS的疾病进展 TB accelerates the progression of HIV disease TB和 HIV 双重感染 TB and HIV co-infection TB是 PLWHA人群中的主要致死原因 TB is the leading cause of death in people living with HIV chest radiography findings consistent with tuberculosis; and lack of response to a trial of broad-spectrum antimicrobial agents. (NOTE: Because the fl uoroquinolones are active against M. tuberculosis complex and, thus, may cause transient improvement in persons with tuberculosis, they should be avoided.) For such patients, if facilities for culture are available, sputum cultures should be obtained. In persons with known or suspected HIV infection, the diagnostic evaluation should be expedited. 诊 断 Diagnosis 标准 6. 对有症状的涂阴儿童胸腔结核患者的诊断应该根据胸部影象学检查 可疑肺结核、暴露史或结核感染（ TST阳性或 干扰素释放试验）。对这样 的病人如果条件允许，应该通过（排出物、洗胃或引痰等方式）得到痰标 本进行培养。 STANDARD 6. The diagnosis of intrathoracic (i.e., pulmonary, pleural, and mediastinal or hilar lymph node) tuberculosis in symptomatic children with negative sputum smears should be based on the finding of chest radiographic abnormalities consistent with tuberculosis and either a history of exposure to an infectious case or evidence of tuberculosis infection (positive tuberculin skin test or interferon gamma release assay). For such patients, if facilities for culture are available, sputum specimens should be obtained (by expectoration, gastric washings, or induced sputum)for culture. 诊 断 Diagnosis 当前可得的结核病诊断方法 What methods are available now to detect TB 涂片显微镜检查 (Microscopy) ZN FM 培养 (Culture) Conventional media MGIT BACT -3D ALERT ESP etc. 核酸扩增法 （ NAAT） Amplicor MTB、 MTD Gen-Probe、 SDA Quanti-PCR Genechip 血清学方法 (Serological method) QuantiFERON T-SPOT TB ELISA etc. 其他 (Other) FAST-Plaque(phage) HPLC etc. 传统的结核病实验室检查方法 # Capacity to detect when 10,000 bacilli per ml sputum * Capacity to detect when 102-3 CFU per ml sputum ZN FM Solid Liquid PPD Sensitivity 40-60% 50-70% 85% 70-85% ? Timing 48hours 48hours up to 8 weeks 2weeks 72hours Cost cheap expensive cheap expensive cheap Stain # Culture* TST 真空采血管使用指南 1.8, 2.4, 4.0, 5.0抗凝剂 :枸橼酸钠与血样比为1:4采血后立即颠倒混匀 8次 试验前混匀标本全血血细胞沉降率试验 Black 黑色 1.8, 2.0, 2.7, 4.5抗凝剂 :枸橼酸钠与血样比为1:9采血后立即颠倒混匀 8次 试验前混匀标本全血血液凝固试验 Lt Bule 浅兰色 2.0, 2.5, 3.0,3.5, 4.0, 5.0, 6.0, 10.0抗凝剂 :K2EDTA或 K3EDTA 采血后立即颠倒混匀 8次 试验前混匀标本全血 血液学 常规试验 Lavender 紫色 5.0抗凝剂 K2EDTA 和惰性分离胶采血后立即颠倒混匀 8次室温离心 1100g， 10分钟血浆分子生物学诊断 White 白色 2.5, 3.5, 4.0, 5.0, 6.0, 9.5, 11.0,13.0惰性胶体促凝剂 采血后立即颠倒混匀 5次 静置 30分钟 离心 血清 快速血清 分离生化 Gold 金黄色管 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 10.0抗凝剂 :肝素钠、锂、氨 采血后立即颠倒混匀 8次 离心血浆 快速血浆 生化 Green 绿色 3.0, 5.3, 7.0促凝剂：纤维蛋白酶采血后立即颠倒混匀 8次 静置 5分钟 离心血清 快速血清 生化 Orange 桔红色 2.0, 3.0, 4.0, 5.0, 7.0, 10.0,15.0,20.0无（内壁涂有硅酮） 采血后不需颠倒混匀 静置 1小时 离心 血清血清生化 Red 红色 采血量 (ml)添 加 剂制 备 步 骤 标本类 型临床用途 管 盖 颜 色 痰涂片显微镜检查 Z-N 萋尼二氏染色 , 需要 5,000-10,000 个 CFU/毫升 100x 油镜 看完 300个视野大 概需要 15分钟 40-70% 灵敏度 , 90%特异 性 FM 荧光染色 灵敏度较 ZN染色高 10% Uneven Too thick Sloughed off Underdecolorized Good Smear Poor Smear 萋尼氏染色 不同痰标本呈现的物理性状 唾液 脓痰 血痰 粘液痰（水样） Culture of the organisms Liquefaction, decontamination, concentration Soild media- Egg-based (Lowenstein- Jensen), agar-based (Middlebrook 7H10 and 7H11), biphasic media Liquid media- Middlebrook 7H9, 7H12 Identification by biochemical tests The entire process: 4 to 6 weeks Drug susceptibility test: add 3 to 6 weeks BACTEC TB system Tubes with a fluorescent compound in silicone bottom, which is sensitive to oxygen presence Comparison of the BACTEC MGIT 960 with Lowenstein-Jensen medium (1-2w vs 6-8w) The recovery rates- 94% (117/124) vs 75.8% (94/124); TTDs (time to detection) - 10.7 vs 30.6 d Disadvantage- inability to observe colonial morpgology, mixture culture, high cost Chien HP et al, Int J Tuberc Lung Dis, 2000 Serodiagnosis of Tuberculosis Antigen 60 IgG (%) IgM (%) Active PTB (n=147) 76.2 9.52 Active EPTB (n=90) 58.9 10 Inactive TB (n=153) 18.3 1.6 Non TB dz (n=517) 9.7 0.6 Sensitivity 69.6 10.5 Specificity 92.1 99.4 Positive predicted v 67.9 Negative predicted v 89.2 Luh KT, Yu CJ, Respirology 1996 Method Sensitivity Specificity Predictive value (%) Accuracy (%) (%) Positive Negative (%) COBAS AMPLICOR 80.6 100 100 98.6 98.6 Culture 86.1 100 100 99.0 99.0 Microscopy 36.1 99.4 95.4 65.0 94.2 Polymerase chain reaction (PCR) Jan IS et al, J Formos Med Assoc 1998 Factors Contributing to the Increase in TB Cases HIV epidemic Increased immigration from high-prevalence countries Transmission of TB in congregate settings (e.g., correctional facilities, long-term care) Deterioration of the public health care infrastructure Transmission and Pathogenesis of TB Caused by Mycobacterium tuberculosis (M. tuberculosis) Spread person to person through airborne particles that contain M. tuberculosis, called droplet nuclei Transmission occurs when an infectious person coughs, sneezes, laughs, or sings Prolonged contact needed for transmission 10% of infected persons will develop TB disease at some point in their lives Sites of TB Disease Pulmonary TB occurs in the lungs 85% of all TB cases are pulmonary Extrapulmonary TB occurs in places other than the lungs, including the: Larynx Lymph nodes Pleura (membrane surrounding each lung) Brain and spine Kidneys Bones and joints Miliary TB occurs when tubercle bacilli enter the bloodstream and are carried to all parts of the body 不是每个暴露在传染源中的人都会被传染 Probability of transmission depends on: Infectiousness Type of environment Length of exposure 10% of infected persons will develop TB disease at some point in their lives 潜伏性感染和结核病 潜伏性感染 结 核病 结 核分枝杆菌存在于体内 结 核菌素 试验 通常 为 阳性 胸部 x-线 检查 通常无异常 胸部 x-线检查 通常异常 痰 涂片、培养阴性 痰涂片、培养阳性 无 症状 有症状（咳嗽、 发热 、体重减少 ） 没有 传 染性 通常在治 疗 之前有 传 染性 不是 结 核病病人 结 核病患者 全球采取了有利措施来遏制结核病 结核病在人类历史上 肆虐超过 3000年 Sources: DeAngelis CD, Flanagin A. Tuberculosis a global problem requiring a gl