微生物感染的实验诊断.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,What is a Diagnosis?,医学的理念：,诊断的理念：,Evolvement of Medical Mode,Medical mode,Biomedical,Biopsychosocial,Concept,Remove of diseases in Biology,Maintenance of life quality in Biology,psychology,and sociology,Focus,Treatment,Prevention:integration of PreventionDiagnosis,Treatment and rehabilitation,Distribution of Social resource,Central-hospital-directed,Community networks of medical service,Method,Experience-based,Evidence-based,Biopsychosocial mode,预防,I 病因控制,减缓发生,减少危害,预防性诊断：,监测,早期诊断：早发现,康复性诊断：功能,治疗性诊断：病因、药物、,治疗,康复,健康诊断：预测、监控,Categorization,of,Diagnosis,按诊断目的分类：,健康性诊断（Healthy diagnosis）,预防性诊断（preventive diagnosis）,治疗性诊断（therapeutic diagnosis）,按诊断方法分类:,物理学诊断（physical diagnosis）,生理学诊断（physiologic diagnosis）,影像学诊断（imageologic diagnosis）,病理学诊断（pathologic diagnosis）,实验室诊断（laboratory diagnosis）,按诊断信息种类：,生物学诊断（Biological diagnosis）,心理学诊断（psychological diagnosis),社会学诊断（sociological diagnosis）,诊断,的目的提供哪些信息？,生理状态信息,预测疾病发生及条件,确定病因,制定治疗方案,追踪治疗效果,药物监测,判断预后,制定康复方案,其它,预防,治疗,康复,医学的内容,遗传易感性,病因（病原微生物）,治疗药物（药敏试验）,追踪疗效,药物监测,判断预后,制定康复方案,其它,诊断,感染的诊断,医学诊断的基本原则,预测与推测判断的重要性：假设是进行检查的必要前提,逐步检查与分析：每次检查方法的选定与结果的分析判断均依赖于上一次的检查方法与结果,综合各种因素进行分析判断：单一指标得出的判断要慎重,Basic Principles of Medical Diagnosis,-3 elements of medical laboratory science,Indicators-,presentational phenomena of diseases:core(golden),peripheral,associated Indicators according to the significance such as Symptomic and signic,physiologic,iconographical,histopathological,cytological,biochemical,molecular biological changes,etc,Methods:,sensitivity,specificity,simplicity,speediness,Explanation,-intrinsic mechanisms of diseases:Status of human body,Etiological factors,Process of evolvement,Basic Principles of Medical Laboratory Diagnosis,-,-Selection of Indicators,Specificity of indicators:,core(golden)and peripheral,direct and indirect,specific and non-specific,Detectability of indicators:,Combination of multiple indicators,Basic strategies of Laboratory Diagnosis for Infectious Diseases,On detecting targets,Pathogenic Diagnosis,Serologic Diagnosis,On diagnostic aims,Primary diagnosis,Confirming diagnosis,Complementary diagnosis,On needed,time,General diagnosis,Rapid diagnosis,微生物学诊断的基本策略 感染性疾病的标记,病原学诊断,（病原微生物相关标记）,活病原体（分离培养与鉴定）,病原体（形态学检查）,相关产物 Ag检测（免疫学技术）,核酸检测（分子生物学技术）,其他成分检测：酶、特殊成分,血清学诊断,-特异性Ab的检测,机体反应标记,非特异性反应标记：症状体征、生理生化指标、免疫学指标,特异性反应标记：Ab的检测,微生物分离培养与鉴定,基本原理：Kochs Postulates,Golden standard for the diagnosis of infectious diseases,Disadvantages:程序繁琐，费时费力,Development trends：automatic cultivation system,Procedure of microbial isolation culture and identification,Specimen,Isolation culture,Identification,Drug sensitivity test,Supplementary test,将病原微生物从病灶中分离出来，获得纯培养,确定病原微生物的分类学地位：科属、种、群、型,筛选敏感药物,形态学检查,利用不同微生物或其感染病灶的形态学特征的差异对其进行识别、确认或区别,不同标本以及不同微生物采用不同技术方法,简便快速，但可靠性不高，通常不作为独立检查方法,Ag检测（免疫学技术）,检测微生物的Ag来确定其种属,简便快速，可靠性较高，可作为独立检查方法，但应注意非特异性反应,IFA：胞内微生物Ag,ELISA：游离微生物Ag,其他：RIA、凝集试验、沉淀试验、补体结合试验、病毒血球凝集试验等,蛋白质芯片：高通量检测,核酸检测（分子生物学技术）,检测微生物的核酸来确定其种属,简便快速，可靠性较高，可作为独立检查方法，但应注意非特异性反应,DNA：PCRSouthern blotting,RNA:RT-PCR、Northern blotting,基因芯片：高通量检测,其他成分检测：,酶试验：快速脲酶试验（HP）、,特殊成分检测：真菌多糖，细菌LPS、胞壁酸、吡啶二羧酸(DAP芽抱)、分枝菌酸（分枝杆菌属、诺卡氏菌属、棒状杆菌属和红球菌属等）。,Regularities of antibody responses and,serologic diagnosis,Ag1,Ag2,2-16w,IgG1,IgG2,潜伏期,症状期,恢复期,康复期,Titer of Abs,IgM1,IgM2,Serologic procedures,Detection of specific IgM by single serum for early diagnosis of primary infections,Detection of total specific Abs or IgG by paired sera for retrospective diagnosis:,antibody titer in the convalescent phase serum sample is at least 4-fold higher than the titer in the acute-phase serum sample,the patient is considered to be infected,Methods Of Serologic Detection,ELISA/RIA,IFA/,EIA,Complement fixation assay,Hemagglutination inhibition,Neutralization,Latex agglutination,Western blotting,Protein chip,Basic Principles of Medical Diagnosis,-,Methods,Sensitivity:最小检出量,Specificity:真实性,Simplicity:简便实用,Speediness：快速,Selection of Strategies and methods,Purposes of diagnosis,Suspected pathogens,Type of Specimens,State of patient:in biopsychosocial medical model,诊断技术方法的发展,单一指标检测：ELISA、IFA等,多种指标联合检测：,多种指标同时检测：Northern blot、Western blot等技术,高通量检测（基因组或蛋白质组检测）：Biochip 技术,DNA芯片,蛋白质芯片,Procedure of Westhern blot,Labeling Immunoassay,Extraction,of Proteins,SDS-PAGE,Blotting,Protein marker,例：SLE抗核抗体普检测,Procedure of Southern blot,Restriction enzyme Digestion,Probe DNA,Labeling,Hybridization,DNA Extraction,Electroph-oresis,Blotting,Agarose,Membrane,Denaturation,Denaturation,例：肝活检细胞中整合HBV DNA 检测,Procedure of Northern blot,Probe preparation,labeling,Hybridization,RNA extract,电泳,Blotting,变性,Agarose,Membrane,Basic Principles of Medical Laboratory Diagnosis,-,-explanation of results,Summarization of all factors including clinic and other indicators based on the examination,Communication between Lab and clinic,Recommendation of further inspects,药物敏感试验,标本,直接涂片镜检,免疫学试验,分子生物学技术,其他检测技术,分离培养,形态学,试验,生化试验,免疫学试验,动物试验,明确诊断,初步诊断,血清学诊断,感染的实验诊断程序,菌落鉴定,辅助诊断,避免杂菌污染,治疗前采取,不同期采取不同部位标本,采集部位病变明显,注意运送保存,微生物学检查标本的采集和运送原则,临床因素,实验室因素,根据检查方法选取不同标本,标本必须新鲜,运送注意保存,Transportation and preservation,To be detected as soon as possible,Low temperature:4 for a temporary preservation and 70 for a long-term preservation,Proper transfer Media:,Broth for bacteria,Buffered glycerol saline for viruses,第一节 细菌学诊断,细菌学检查标本的采集与送检,用于分离培养的标本：,血清学诊断的标本：,采取急性期、,防止细菌死亡,双份血清,避免杂菌污染,用抗菌素以前,不同期不同标本,标本必须新鲜,运送注意保存采集病变明显部位,细菌学检查标本的采集和运送,临床因素,实验室因素,检查方法,标本必须新鲜,运送注意保存,药物敏感试验,标本,直接涂片镜检,分离培养与鉴定,生化试验,免疫学试验,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,血清学诊断,形态学鉴定,细菌感菌检查程序,菌落鉴定,不染色标本的检查：细菌动力,单染色法：细菌形态,复染色法：细菌形态、结构与鉴别,革,兰,染色法（,Gram stain,）,抗酸染色法（,acid-fast stain,）,特殊染色法：负染色、鞭毛染色,细菌的形态学检查法,Light microscope,Standard light microscope,Dark-field microscope,Phase-contrast microscope,fluorescence microscope,Confocal scanning microscope,Electron microscope,Transmission electronic microscope,Scanning electronic microscope,Microscopy,革兰染色法,（Gram stain）,方法：,1、结晶紫初染 1 min,2、卢戈氏碘液媒染 1 min,3、95酒精脱色 30sec1 min,4、稀释石炭酸复红复染 1 min,结果：,紫色G,红色G,革兰染色的原理和意义,原理：,G+等电点低，带正电荷的碱性燃料作色牢固,G+细胞壁肽聚糖层多，G-细胞壁脂质含量高,G+含有多量核糖核酸镁盐与结晶紫、碘结合成大分子复合物,意义：,鉴定细菌,与致病性有关,参考选择抗菌药物,方法：,1、石炭酸复红初染：加热35min，水洗,2、3%5%盐酸酒精脱色30s，水洗,3、碱性美蓝复染30s，水洗。,4、干后镜检,结果：,抗酸杆菌红色 非抗酸杆菌蓝色,原理：分枝杆菌属的细菌，胞壁脂质多，其中的分枝菌酸与石炭酸复红结合成牢固的复合物，能抵抗脱色,医学意义：鉴定细菌,抗酸染色,（acid-fast stain）,Direct microscopic examination,N.Meningococcus in cerebrospinal fluid,-hemolytic streptococcus in blood,Procedure of bacterial isolation culture and identification,Isolation culture,Colony observation,Morphological observation,Biochemical test,Virulence test,Immunological test(typing),Microphage test(typing),Animal test,Molecular biological test,Specimen,Agar plate,Pure culture,Identification,Drug sensitivity test,Methods of bacterial identification,Morphological:,shape,arrangement,size,staining,structure,Physiological:,characteristics in nutrition,Biochemical test,growth,Aerobic vs.anaerobic,colony appearance(smooth,rough,mucus type,hemolytic,pigment),Immunological:,antigenicity,Molecular biological:,analysis of DNA,RNA,protein,Virulence:,test for toxin production,Animal test,对,糖,的,发,酵,对,蛋白质的发酵,枸橼酸盐利用试验,尿素酶试验,甲基红试验,吲哚试验,硫化氢试验,单糖发酵试验,VP试验,其他试验,常见的生化反应,标本：,双份血清标本，恢复期血清中抗体效价比急性期，血清中抗体效价升高4倍者方有意义,种类,原理：,用已知的细菌或其特异性抗原检测患者体液中有无相应特异性抗体和其效价的动态变化，可作为某些传染病的辅助诊断。,凝集试验：,沉淀试验：,补体结合试验：,中和试验：,肥达试验、外斐试验,梅毒VDRL、RPR试验等,抗链“O”试验等,Q热柯克斯体抗体检测等,血清学诊断,Serologic Diagnosis of bacterial infection,Detection of Antibodies against the infectious agents,Methods:agglutination test,precipitation test,complement fixation test,neutralization test,ELASA,Widal test:agglutination test to detect Abes against Salmonella,Antistreptolysin O(ASO)test:a neutralization test to detect Abes against hemolysin O secreted by,-hemolysis,Streptococcus,Antistreptolysin O(ASO)test,A neutralization test to detect Abs against hemolysin O secreted by,-hemolysis,Streptococcus for post-infectious diagnosis e.g.Rheumatic arthritis and glomerulonephritis,1:250+;1:400 active,Widal test-,retrospective serodiagnosis,Agglutination test in test tubes,to detect Abs against Salmonella for complementary diagnosis of Typhoid fever,O antigen(,common antigen),:,1:80,early,phase,H antigen:,1:160,for determining the species,Serum,1:20,1:40,1:80,1:160,1:320,NS,O,antigen,0.5ml,0.5ml,0.5ml,0.5ml,0.5ml,0.5ml,H,of,Typhi,0.5ml,0.5ml,0.5ml,0.5ml,0.5ml,0.5ml,H,of,paratyphi A,0.5ml,0.5ml,0.5ml,0.5ml,0.5ml,0.5ml,H,of,paratyphi B,0.5ml,0.5ml,0.5ml,0.5ml,0.5ml,0.5ml,0.5ml 37,O/N，endpoint,Widal test,动物实验,细菌分离培养,测定细菌毒力,制备免疫血清,建立动物模型,快速诊断,Direct microscopic examination,Antigen detection,DNA or RNA detection,自动化检测系统,诱因：,破坏细胞壁的物质,症状：,慢性为主，症状不典型,培养：,高渗培养基（油煎蛋样菌落）,镜检：,多型性,L型细菌的诊断,药敏试验,琼脂纸片扩散法（K-B法）,稀释法:最低抑菌浓度（MIC),E-test,Susceptibility test,Tube dilution method,Minimal inhibitory concentration(MIC):the smallest amount of chemotherapeutic agent required to inhibit the growth of organism in vitro,Disk diffusion method,Zone of inhibition(ZOI):the correlation of ZOI and MIC has been established by FAD,E Test.This commercially-prepared strip creates a gradient of antibiotic concentration when placed on an agar plate,病毒感染的诊断,标本,直接形态学检查,分离培养与鉴定,免疫学试验,动物试验,分子生物学技术,其他检测技术,血清学诊断,形态学鉴定,病毒的检查程序,标本采集与送检,用于分离培养的标本：,血清学诊断的标本：,采取急性期、,防止病毒灭活,双份血清,病毒的形态学检查,细胞病变(Cytopathic effect,CPE),包涵体（Inclusion Bodies）,电镜观察,巨细胞病毒包涵体,动物接种,鸡胚接种,组织培养,器官培养,移植培养,细胞培养,原代细胞培养,二倍体细胞培养,传代细胞培养,三大培养方法,常规分离与鉴定,病毒的培养,动物（Animals）,鸡胚（Embryonated eggs）,组织培养（Organ and tissue culture）,Organ culture,胚胎Embryo culture,Cell culture:原代细胞,传代细胞,双倍体细胞,常用的细胞培养,1、CPE,2、红细胞吸附,正常细胞,病变细胞,病毒在细胞内增殖的指标,Observation of CPE,Hemadsorption test,Interference test,Viral antigen detection-Labeled immunostaining,Viral detection in cell culture,CPE of HSV,Syncytia of CMV,Normal HEK cell,inclusion bodies of babies virus,Cytopathic effects:cell rounding due to HSV;syncytium formation or cell fusion due to RSV in human embryo fibroblast cells,Hemadsorption test,Glycoproteins(hemagglutinin,HI)of virus can adsorb erythrocytes of animals,e.g.influenza virus,Interference test,Indicates exist of target virus by interfering virus with CPE or hemadsorption,CMV early antigen demonstrated in nuclei of human embryo fibroblasts by immunofluorescence assay(IFA),The envelope glycoprotein I in VZV labeled with McAb and anti-mouse IgG conjugated with 15 nm colloidal gold,免疫扩散,中和试验,补体结合试验,血凝抑制试验,标记免疫技术,病毒的血清学诊断,病毒的快速诊断,光镜-,包涵体,电镜-,病毒颗粒,免疫荧光-,Ag,ELISA-Ab,分子生物学技术,杂交,Southern blot,PCR,Northern blot,biochip,Detection of viral genetic material,PCR(Polymerase chain reaction)or RT-PCR(Reverse transcriptase polymerase chain reaction),Southern（DNA),Northern(RNA),and dot blotting,DNA genome hybridization in situ(cytochemistry),Electrophoretic mobilities of RNA for segmented RNA viruses(Electrophoresis),Restriction endonuclease cleavage patterns,第三节 真菌学诊断,标本,直接形态学检查,分离培养与鉴定,免疫学试验,动物试验,分子生物学技术,其他检测技术,血清学诊断,形态学鉴定,真菌的检查程序,菌落鉴定,标本,浅部感染真菌取毛发等标本,深部感染真菌取痰、血液、脑脊液等,直接镜检：,标本（,皮屑,、毛发）10%KOH,假丝酵母菌-取材涂片，革兰染色,隐球菌-,脑脊液,离心，沉淀涂片，墨汁负染,结果判断：,阴性不能排除感染。,组织中分隔分枝的菌丝为曲霉;粗大、不分隔或不分枝的菌丝为毛霉。,皮肤癣菌菌丝肥大粗长为处于活跃状态。,酵母型和二相型真菌在组织内常为孢子。,表皮癣菌的检出,新生隐球菌脑脊液涂片,Sabouraud 培养基,快速诊断,血清学诊断,核酸的诊断,毒素的检测,分离培养与鉴定,鉴定,菌落观察,形态学观察,Comparison of diagnostic strategy of 3 kinds of microorganisms,virus,Bacterium,Fungus,Isolation and identification,+,+,+,Microscopic examination,+,+,+,Detection of antigens,+,+,Detection of Nuclear acids,+,Detection of IgM by single serum,+,Detection of IgG by paired sera,+,+commonly used,+used,occasionally used,seldom used,复习要点,概念：病原学诊断、血清学诊断、分离培养、细菌鉴定、药物敏感试验、K-B法、E-test、MIC、细胞培养、血细胞吸附FIA、病毒血凝抑制试验、,微生物学检查标本采集的基本原则,微生物学检查的基本策略和方法及其基本原理,细菌病原学检查的基本程序和方法,病毒分离培养与鉴定的方法,真菌感染微生物学检查的常用方法,复习思考题,微生物学检查所用标本采集的基本原则是什么？,微生物学检查的基本策略有哪些？,微生物学检查的基本方法有哪些？其基本原理是什么？,细菌、真菌和病毒性疾病的实验诊断策略有何不同？为什么？,微生物学检查常用方法有哪些？它们在细菌、真菌和病毒性疾病的实验诊断中有何相同点和不同点？,你对感染性疾病诊断的内涵和价值是如何理解的？你认为循证医学理念下的感染性疾病诊断与以往有何不同？,课外阅读书目,Hawley LB，微生物与感染性疾病，中信出版社，2004,闻玉梅，现代医学微生物学，上海医科大学出版社，1999,Brooks GF，Medical Microbiology，人民卫生出版社，2001,蔡文成,实用临床微生物诊断学，东南大学出版社，1998,杨占秋,诊断和实验病毒学，郑州大学出版社，2002,Thank you,