病原学诊疗和防治.ppt

2019/9/23,1,第一篇 微生物学基础,第八章 病原学诊断与防治 Laboratory Diagnosis and Prevention Principles 第一节 细菌学诊断 第二节 病毒学诊断 第三节 真菌学诊断 第四节 特异性预防与治疗,2019/9/23,2,Outline,Laboratory diagnosis for bacterial infections Morphological - microscopic Molecular Serological Laboratory diagnosis for viral infections Morphological - electron microscopic Isolation Molecular: nucleic acids and proteins Serological Laboratory diagnosis for fungal infections Morphological - microscopic Isolation,2019/9/23,3,2019/9/23,4,Assays,Morphological assays Light or electron microscopies Isolation and differentiation Serological assays Antigen-antibody assays Molecular assays Microorganisms gene (DNA & RNA),2019/9/23,5,Principles for Specimen Collection,Sterile manipulation, avoid contamination Obtain a specimen from the infected site according to the disease phase before administrating antibiotics Double sera for antibody detections, acute and recovery phase each Positive if the titer of the later specimen is above 4X to the first specimen Transport, and store correctly as soon as possible Store at -4oC for most bacterial specimens, but keep warm for Neisseria meningitidis and Neisseria gonorrhoeae Store at -4oC -70oC for viral specimens,2019/9/23,6,Laboratory Diagnosis for Bacterial Infections,Morphological Protein and Nucleic acids Antibody,2019/9/23,7,标本的采集和送检六原则,无菌取材,避免杂菌污染,不同期不同标本,用抗生素以前,采集病变明显部位,运送注意保存,标本必须新鲜,2019/9/23,8,病原菌的检查程序,药物敏感试验,标本,直接涂片镜检,分离培养,生化试验,血清学试验,动物试验,明确诊断,初步诊断,分子生物学技术,其他检测技术,2019/9/23,9,Bacteriological Diagnosis,Identifying the organism Morphological Isolation & culture Biochemical reaction Serological assays,Pathogenesis & antibiotics susceptibility Animal experiment Virulence test antibiotics susceptibility,2019/9/23,10,Morphological,Non-stained microscopic observation Dark-field microscopy Observing the movement of live bacteria Stained microscopic observations Gram stain Acid-fast stain Fluorescence stain,2019/9/23,11,2019/9/23,12,2019/9/23,13,2019/9/23,14,2019/9/23,15,2019/9/23,16,2019/9/23,17,Isolation & Culture: Colony,Size Shape Color Surface features Smooth - Rough Transparency Hemolysis,2019/9/23,18,2019/9/23,19,2019/9/23,20,分离培养 isolation and cultivation,分离培养是微生物学诊断的金标准，是最可靠的传统诊断技术。 （图为普通培养箱）,2019/9/23,21,厌氧罐 厌氧袋,2019/9/23,22,Biochemical Reactions,Sugar Fermentation H2S Test Citrate utilization,2019/9/23,23,Serological Assays,Detection antibody in the patients serum A current infection should be IgM positive A 4-fold or greater rise on antibody titer between the acute serum sample and the convalescent serum sample Major drawbacks A single IgG antibody titer is difficult to interpret because it is unclear whether it represents a current or a previous infection the convalescent sample is usually taken 10-14 days after the acute sample. By this time, the patient has often recovered and the diagnosis becomes a retrospective one Some exceptions In certain diseases, a single titer of sufficient magnitude can be used as presumptive evidence of a current infection,2019/9/23,24,基本原则 用已知抗体检测未知抗原； 用已知抗原检测未知抗体。 最简易的方法有玻片凝集等试验。 常用的方法有：凝集试验、沉淀试验、补体结合试验、ELISA、免疫荧光等。,免疫学试验,2019/9/23,25,Animal experiment,Animals Mouse Guinea Pig Rabbit Dog Monkey Inoculation routes Intradermal Subcutaneous Intraperitoneal Intravenous Intracranial / intracerebral Intraspinal Intranasal Lavage,2019/9/23,26,Virulence Test,Median lethal dose, LD50 Median infective dose, ID50 Elek Plate Diphtherotoxin Corynebacterium diphtheriae Enterotoxin enterotoxigenic E. coli,2019/9/23,27,Antibiotic Susceptibility Test,Method,2019/9/23,28,MIC & MBC,Minimum Inhibitory Concentration，MIC Minimum Bactericidal Concentration，MBC Bactericidal drugs usually have an MBC equal or very similar to the MIC Bacteriostatic drugs usually have an MBC significantly higher than the MIC,2019/9/23,29,2019/9/23,30,Bacterial Proteins, DNA & RNA,Antigens (Proteins) Known antibodies Agglutination, coagulation, precipitation, ELISA, Immunofluorescence, radioimmunoassay DNA & RNA PCR Nucleic acid hybridization,2019/9/23,31,Detection of Bacterial Antigens,Precipitation test Coagglutination test Immunoflorecence, IF McAb technique,2019/9/23,32,2019/9/23,33,2019/9/23,34,Bacterial DNA & RNA,PCR DNA probe & hybridization DNA hybridization (southern blot) Plasmid fingerprint analysis Restrict multimorphologic analysis,2019/9/23,35,2019/9/23,36,2019/9/23,37,PCR,2019/9/23,38,分子生物学技术 molecular biological techniques,核酸杂交 nucleotide hybridization 原位杂交 in situ hybridization 斑点杂交 dot blot Southern blot Northern blot PCR 技术：多聚酶链反应 (polymerase chain reaction，PCR ),2019/9/23,39,PCR 是一种无细胞的分子克隆 技术，能在体外将一个基因或 一段DNA 序列经过多次循 环的DNA复制，在数小时内扩 增上百万倍。既便于基因诊断，又利于 研究。,2019/9/23,40,2019/9/23,41,Gene Chip / microarray,2019/9/23,42,Diagnosis Strategy for Bacterial Infection,2019/9/23,43,第25章 病毒感染的诊断 P238 Laboratory Diagnosis for Viral Infection,Morphological Isolation & Culture Protein & Nucleic acids Serological,2019/9/23,44,病毒感染的检测,一、标本的采集与运送,急性期标本； 根据感染性质取不同的标本； 标本置于转运液中； 冷藏快运。,2019/9/23,45,Morphological,Electron microscopy, EM Immunoelectron microscopy, IEM Light Microscopy Inclusion body Negri body,2019/9/23,46,2019/9/23,47,二、病毒分离培养 病毒分离的一般程序是,接种易感动物 出现症状 血清学 检测标本 杀灭杂菌 鸡胚 病变或死亡 鉴定 细胞培养 细胞病变,2019/9/23,48,Isolation and Culture,Animal inoculation Chicken egg culture: 914 days Tissue culture Organ culture Tissue culture Cell culture,2019/9/23,49,Cell culture,2019/9/23,50,2019/9/23,51,Type of Cultured Cells,Primary cell culture Passage 2-3 generations Diploid cell culture 2-fold chromosomes, Passage 50100 generations, safe, can be used for vaccine manufacture Continuous cell line culture Mutant diploid cells, mostly tumor cells, easy to passage and maintain Not sensitive as diploid cells, may have endogenous viruses, can not be used for vaccine manufacture,2019/9/23,52,Indications of Viral Propagation,Cytopathic effect, CPE Hemadsorption Hemagglutination Viral interference Neutralization test, NT,2019/9/23,53,2019/9/23,54,2019/9/23,55,（1）原代细胞培养： 动物或人组织直接用蛋白酶消化所得的细胞 经体外培养后可贴壁或悬浮生长。 （2）二倍体细胞株： 在体外连续培养5060代仍保持其二倍染色体数目的单型细胞。,为生产人用疫苗的首选细胞株。,（3）传代细胞系 能在体外连续传代、来自癌细胞或发生自发转 化、突变的原代细胞或二倍体细胞株。,2019/9/23,56,临床病毒实验室常用的细胞培养,2019/9/23,57,识别病毒增殖的方法 （1）细胞病变效应（CPE）：溶细胞型病毒感染细胞后，可出现细胞团缩、裂解或细胞肿大、数个细胞融合成多核巨细胞或细胞聚集成葡萄串状、脱落或死亡等。,2019/9/23,58,epithelial cells - adenovirus,uninfected,early infection,late infection,2019/9/23,59,Negri Body,2019/9/23,60,（2）红细胞吸附：流感病毒和某些副粘病毒感染细胞后2448小时，细胞膜上出现病毒的血凝素，能吸附豚鼠、鸡等动物及人的红细胞，发生红细胞吸附现象。 （3）干扰现象,2019/9/23,61,病毒量测定 （1）测定病毒数量可用空斑形成单位（PFU）表示。一个空斑是标本中一个病毒大量复制所致，有感染性的病毒经适当稀释后接种于单层细胞，由于单个病毒的复制增殖使局部单层细胞脱落，此即空斑。经染色后空斑可用肉眼观察，计算空斑数即可计数病毒数量。,2019/9/23,62,PLAQUE ASSAY,PLAQUE ASSAY,2019/9/23,63,PLAQUE ASSAY,PLAQUE ASSAY,2019/9/23,64,2019/9/23,65,三、光学显微镜和电子显微镜 1、光学显微镜：观察病毒的包涵体,2019/9/23,66,2、电子显微镜：直接观察病毒,2019/9/23,67,Viral Quantity Determination,TCID50 50% tissue culture infectious dose Determine the infectivity of the examined viruses PFU Plaque formation pfu/ml Precisely quantitate the number of the living viral particles,2019/9/23,68,2019/9/23,69,Plaque,2019/9/23,70,Differences between viral particle (VP), plaque formation unit (PFU),Viral particles (VPs) represent the total number of viral particles (live and dead combined). Due to variations in virus preparations the ratio of live/dead varies significantly and therefore, VP does not reflect the amount of active virus in the preparation PFU (plaque formation unit) represents the number of infectious or live viruses. It reflects the amount of working viruses in the preparation For most virus preps, the VP/PFU ratio is 20:1 to 50:1 Which one better reflects the amount of active virus used? Using VP (viral particles) as the unit will result in significant variations in the amount of actual live viruses used, and using IFU or PFU as the viral unit will give more consistent outcomes.,2019/9/23,71,Proteins and Nucleic Acids,Proteins (antigens) Fluoresece-labeled, enzyme-labeled (enzyme linked immunosorbent assay, ELISA) and radio-labeled anitbodies Viral DNA or RNA Gene chip DNA chip / cDNA Microarray,2019/9/23,72,Virus-specific Antibodies,Neutralization test, NT Hemagglutination inhibition test, HI Complement fixation, CF ELISA Western blot,2019/9/23,73,三、病毒感染的血清学诊断方法 P240 1、中和试验 2、补体结合试验 3、红细胞凝集和凝集抑制试验,病毒抗体 失去感染性,恢复期/早期 4:1，有诊断意义。,2019/9/23,74,ELISA for HIV antibody,Microplate ELISA for HIV antibody: coloured wells indicate reactivity,2019/9/23,75,2019/9/23,76,Diagnosis Strategy for Viral Infection,2019/9/23,77,Rapid Diagnosis for Viral Infection,Morphological EM & IEM, occasionally Light Microscopy: inclusion body Viral antigens Immunofluorescence Radioimmunoassays ELISA Specific antibodies: IgM Nucleic acids: PCR,2019/9/23,78,小 结,细菌感染的微生物学检查法 病原体检查 核酸的检测 抗原、抗体的检测 病毒感染的微生物学检查法 电镜及免疫电镜 病毒的分离培养和鉴定 核酸的检测 抗原、抗体的检测 真菌感染的微生物学检查法 直接镜检 真菌的分离培养,2019/9/23,79,第四节 特异性预防与治疗 Prevention Principles,2019/9/23,80,特异性免疫的分类,自然免疫 Natural Immunization 自然主动免疫：隐性或显性感染 自然被动免疫：通过胎盘或初乳获得母体抗体 人工免疫 Artificial Immunization 人工主动免疫：接种疫苗或类毒素等 人工被动免疫：注射抗毒素或丙种球蛋白等,2019/9/23,81,一、人工免疫(artificial immunization),用人工的方法给机体输入抗原性物质（如疫苗、类毒素等）或直接输入免疫效应分子（如抗体、细胞因子等），使机体获得特异性免疫力的方法。 人工主动免疫 artificial active immunization 人工被动免疫 artificial passive immunization,2019/9/23,82,人工免疫：采用人工方法将疫苗、类毒素等或含有某种特异性抗体、细胞免疫制剂等接种于人体，以增强宿主体的抗病能力。,人工自动免疫 artificial active immunization 疫苗（vaccine） 类毒素,人工被动免疫 artificial passive immunization 抗毒素 抗菌血清 血清丙种球蛋白,2019/9/23,83,人工自动与被动免疫比较,2019/9/23,84,生物制品(biological products)：人工免疫的免疫原、免疫血清、细胞制剂及诊断制剂的生物性制剂。,人工主动免疫生物制品 疫苗 类毒素 人工被动免疫生物制品 抗毒素 丙种球蛋白,2019/9/23,85,疫苗（vaccine）,减毒活疫苗（attenuated live vaccine） 卡介苗（BCG） 灭活疫苗（inactivated vaccine） 伤寒沙门菌与甲、乙型副伤寒沙门菌混合的三联疫苗 亚单位疫苗（subunit vaccine） 基因工程疫苗（gene engineered vaccine） 核酸疫苗（nucleic acid vaccine）,2019/9/23,86,2019/9/23,87,2019/9/23,88,类毒素（toxoid）,细菌外毒素经0.4甲醛液处理后，其毒性消失而仍保留抗原性的生物制品 常用的类毒素 白喉类毒素 破伤风类毒素等 白、百、破三联疫苗,2019/9/23,89,人工被动免疫生物制品,抗毒素（antitoxin） 通常是用细菌类毒素给马多次注射后，取其免疫血清提取免疫球蛋白精制而成 常用 破伤风抗毒素 白喉抗毒素 免疫球蛋白（immunoglobulin）,2019/9/23,90,人工主动免疫病毒疫苗,减毒活疫苗（attenuated live vaccine） 灭活疫苗（inactivated vaccine） 亚单位疫苗（subunit vaccine） 基因工程疫苗（gene engineered vaccine） 基因工程亚单位疫苗（gene engineered subunit vaccine） 基因工程载体疫苗（gene engineered vectored vaccine） 基因缺失活疫苗（gene deleted live vaccine） 核酸疫苗（nucleic acid vaccine）。 合成肽疫苗（synthetic peptide vaccine） 抗独特型疫苗（anti-idiotype vaccine）,2019/9/23,91,活疫苗（Living vaccine）,减毒活疫苗 基于基因工程技术的新型活疫苗 遗传重组活疫苗 基因缺失活疫苗 载体活疫苗,2019/9/23,92,减毒活疫苗(Attenuated vaccine),自然或人工选择法（如Ts株）对人低毒或无毒的变异株，如脊灰、流感、麻疹的减毒株。 优点：模拟自然感染，免疫效果好，局部IgA。 理论上的缺点，实际工作中少见 变异株可能回复到有毒力的野生株 如果机体免疫缺陷，减毒株仍可能引起感染或并发症 可能激活机体内其他病毒的感染 减毒活苗可能引起持续感染 成功的范例：1961年我国应用脊灰疫苗，脊灰发病率大幅度下降。1980年天花病毒灭绝。,2019/9/23,93,遗传重组疫苗,将野毒株表面抗原基因与弱毒株其他基因组合而获得减毒活病毒。 适用于基因组分节段双链RNA病毒，如流感病毒、轮状病毒等。,2019/9/23,94,基因缺失活疫苗,利用DNA重组技术，定向缺失病毒基因组的某一区段，使病毒丧失毒力而保留增殖能力和免疫原性。 优点：疫苗株的遗传背景清楚，不易突变回到野毒株。,2019/9/23,95,载体活疫苗,利用DNA重组技术，将编码病毒抗原的基因插入到另一无毒力的载体病毒，使之高效表达 易建立多价疫苗 载体病毒常用的有痘苗病毒、腺病毒、HSV-1,2019/9/23,96,灭活疫菌（Killed vaccine）,灭活全病毒疫苗 亚单位疫苗 合成肽病毒疫苗 基因工程疫苗 独特型病毒疫苗 核酸疫苗,2019/9/23,97,灭活全病毒疫苗,物理或化学方法灭活病毒。适用烈性或易变异病毒如乙脑病毒、狂犬病病毒、流感病毒。 优点：生产简单、易保存运输 缺点 需要大量培养病毒，成本高 无局部抗体，不激活CTL，可激活TDTH 甲醛灭活的麻疹病毒和RSV疫苗可加重疾病，机制不详,2019/9/23,98,亚单位疫苗（Subunit）,种类 化学方法制备：如HBsAg和流感HA 基因