第8章-病原学诊断与防治--课件

,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,ppt课件,*,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,ppt课件,*,第八章 病原学诊断与防治,第一节 细菌学诊断,包括；,细菌学诊断,检测病原菌及其抗原，产物或核酸,血清学诊断,检测患者血清中特异性抗体,1,ppt课件,第八章 病原学诊断与防治 第一节 细菌,一、病原菌检测,根据不同疾病以及疾病不同时期，采取不同标本。,标本采集时应注意无菌操作，尽量避免杂菌污染。,应在使用抗菌药物前采集标本，否则注明药物种类。,标本必须新鲜，采集后尽快送检，或冷藏送检。,标本做好标记，详细填写化验单，保证各环节的准确无误。,标本采集与送检原则,2,ppt课件,一、病原菌检测根据不同疾病以及疾病不同时期，采取不同标本。标,致病菌的检验程序,标本,直接涂片染色镜检,分离培养与鉴定,病原菌抗原检测,其它检测,涂片镜检,菌落特征,生化反应,药敏试验,血清学反应,免疫荧光,酶联免疫测定,免疫印迹,细菌代谢产物,（气相色谱）,细菌核酸,（,PCR）,噬菌体分型,动物试验,3,ppt课件,致病菌的检验程序标本直接涂片染色镜检分离培养与鉴定病原菌抗原,Hood and chamber for anaerobic culture,Blue indicate oxygen,4,ppt课件,Hood and chamber for anaerobic,Microscopy and stains,Simple and less sensitive (bacteria No. 10,5,/ml),Gram positive,(Staphylococcus),Gram negative,(E.coli),Acid fast stain,Mycobacterium tuberculosis, From:CDC,5,ppt课件,Microscopy and stainsGram posi,血平板上的,金黄色葡萄球菌菌落（黄色）及,表皮葡萄球菌菌落（白色）,6,ppt课件,血平板上的6ppt课件,梅毒螺旋体,Treponema pallidum,(pathogen of syphilis),直接荧光抗体检测,Direct fluorescent antibody test,(From Morse S et al:,Atlas of sexually transmitted diseases and AIDS,St Louis, 2003, Mosby.),7,ppt课件,梅毒螺旋体Treponema pallidum (patho,二 血清学诊断,用已知的细菌或其特异性抗原检测患者血清或其它体液中特异抗体和其效价的动态变化，可作为某些感染性疾病的辅助诊断。一般采取病人的血清进行试验，故称为,血清学诊断(,serological diagnosis),。,血清学诊断最好取患者急性期和恢复期双份血清标本，当后者的抗体效价比前者,升高大于等于4倍,时方有意义。,恢复期,Convalescent phase,急性期,Acute phase,8,ppt课件,二 血清学诊断恢复期 Convalescent phase急,直接凝集试验,The Fab portions of the,antibodies link microorganism together causing agglutination,on the slide,From: Dr.,Gary E. Kaiser,常用方法：直接凝集试验、乳胶凝集试验、,ELISA。,9,ppt课件,直接凝集试验The Fab portions of the,10,ppt课件,10ppt课,第二节 病毒学诊断,一 标本的采集与送检,早期（前驱期、急性期、发病前12天）,根据不同病毒感染，采集不同部位标本,标本无菌采集与处理（加抗生素）,病毒易被灭活，低温保存，快速送检,血清学实验检查抗体（双份血清）,11,ppt课件,第二节 病毒学诊断 一 标本的采集与送检11ppt课件,二 病毒的分离与鉴定,（,virus isolation and determination）,（一）病毒的分离,动物接种：选择易感动物及接种部位，观察发病情况。,鸡胚培养：不同病毒选择不同接种部位，羊膜腔，分离流感病毒最常用。,细胞培养：分离病毒最常用的方法,（1）原代细胞：来源于动物、鸡胚、人胚组织细胞，对多种病毒敏感，但只能传2-3代。,（2）二倍体细胞：在体外分裂50-100代后仍保持2倍体染色体数目的单层细胞，用于疫苗生产。,（3）,传代细胞：能在体外持续传代的细胞，多由癌细胞或二倍体细胞突变而来。,羊膜腔,Different parts of an egg,embryo, 病毒的分离与鉴定（virus isolation,病毒在培养细胞中增殖的指标,细胞的变化,（1）出现细胞病变效应（,cytopathic effect, CPE）：,即某些病毒在细胞内增殖引起的特有的细胞病变（细胞变圆、聚集、坏死、溶解、脱落等）,Uninfected,CPE,Epithelial cells infected by adenovirus,13,ppt课件,病毒在培养细胞中增殖的指标细胞的变化UninfectedCP,病毒在培养细胞中增殖的指标,细胞的变化,（1）出现细胞病变效应,（2）形成细胞融合（多核巨细胞）,Syncytium formation by,麻疹病毒,measles virus. Multinucleated giant cell (,arrow,) visible in a histologic section of lung biopsy tissue from a measles virus-induced giant cell pneumonia in an immunocompromised child.,14,ppt课件,病毒在培养细胞中增殖的指标细胞的变化Syncytium fo,细胞的变化,（,1）出现细胞病变效应,（2）形成细胞融合（多核巨细胞）,（3）包涵体,Neuron without Negri bodies,内基小体,Negri body in infected neuron,The intracytoplasmic inclusion body in nerve cells is called Negri body, is sign for rabies.,From:CDC,15,ppt课件,细胞的变化Neuron without Negri bodi,Hemadsorption:,attachment of erythrocytes to surface of virus-infected cells, due to the presence of virus-encoded hemagglutinin(influenza, parainfluenza, mumps,togaviruses,) in the cellular membranes.,细胞的变化,红细胞吸附,16,ppt课件,Hemadsorption: attachment,病毒在培养细胞中增殖的指标,细胞的变化,红细胞吸附,干扰现象,细胞代谢的变化,17,ppt课件,病毒在培养细胞中增殖的指标17ppt课件,（二）病毒的数量与感染性测定(嗜斑法）,Plaque assay,Serial dilutions of virus have been plated on confluent monolayer cultures of cells. The cells are stained after a period of time in which a single virus infects a cell, produces new virus particles and infects surrounding cells. The white areas show areas of the culture in which the cells have been killed. Each plaque is the result of the presence of one original infectious virus particle.,From:Dr.M.Hunt,18,ppt课件,（二）病毒的数量与感染性测定(嗜斑法）Plaque assa,(三)新分离病毒的鉴定,核酸类型、理化性状、形态、血清学鉴定、基因测序和生物对比。,19,ppt课件,(三)新分离病毒的鉴定19ppt课件,三、病毒感染的血清学诊断,用已知病毒抗原检测病人血清中相应抗体，以诊断病毒性疾病（中和实验、补体结合实验、血凝抑制实验、凝胶免疫扩散等）,双份血清,20,ppt课件,三、病毒感染的血清学诊断用已知病毒抗原检测病人血清中相应抗体,四、 病毒感染的快速诊断,(一) 形态学,电镜：直接观察病毒,光镜：包涵体,Negative stain of Rabies Virus under EM,From:Wadsworth center, NY Dept of Health,EM of,轮状病毒,rotavirus.,Bar,= 100 nm.,(From Fields BN et al, editors:,Virology,New York, Raven, 1985.),EM of Human immunodeficiency virus. Note the cone-shaped nucleocapsid in several of the virions.,21,ppt课件,四、 病毒感染的快速诊断 (一) 形态学 N,免疫电镜下的甲肝病毒,Hepatitis A virus particles found in fecal extracts by immunoelectron microscopy. Both full and empty particles are present. The virus is 27 to 29 nm in diameter. (X 125,000.),22,ppt课件,免疫电镜下的甲肝病毒Hepatitis A virus pa,（二）病毒蛋白抗原检查,用已知特异性抗体直接检测病毒蛋白抗原（简便、敏感、特异）,常用方法：,免疫荧光技术（,IF）,固相放射免疫测定（,SPRIT）,酶免疫技术（,EIA）,等。,23,ppt课件,（二）病毒蛋白抗原检查23ppt课件,Immunofluorescent assay of,狂犬病毒,rabies virus antigen on animal brain tissue,Positive,Negative,From:CDC,24,ppt课件,Immunofluorescent assay of 狂,（三） 检测病毒特异性,IgM,抗体,检测病毒特异性,IgM,抗体可诊断急性感染， 如风疹病毒。,25,ppt课件,（三） 检测病毒特异性IgM抗体 检,（四） 检测病毒核酸,1 核酸杂交,斑点杂交（,dot hybridization）,原位杂交（,in situ hybridization）,DNA,印迹杂交（,Southern blot）,RNA,印迹杂交 （,Northern blot）,2,核酸扩增,聚合酶链反应 （,polymerase chain reaction，PCR )）,反转录,PCR（Reverse transcription PCR，RT,PCR),3 基因芯片技术（,gene chip）,26,ppt课件,（四） 检测病毒核酸 1 核酸杂交26ppt课件,PCR principle,fig.cox.miami.edu/,27,ppt课件,PCR principle27ppt课件,A,microarray,is a set of short,Expressed Sequence Tags,(,ESTs,) made from a,cDNA,library of a set of known gene loci. The,ESTs,are spotted onto a cover-slip-sized glass plate.,mRNA,are prepared from the tissue of a control organism and an experimental treatment.,Complementary DNA,(,cDNA,) are prepared and labelled with two different fluorescent dyes. The experimental and control libraries are hybridized to the,microarray.,Dual-channel laser excitation excites the corresponding dye, which fluoresces proportional to the degree of hybridization that has occurred. Relative gene expression is measured as the ratio of the two fluorescences: “,up-regulation,” relative to the control will be visualized as a red “pseudo-colour,” “,down-regulation,” shows as green, and,constitutive expression,as a neutral,black,. Intensity of color is proportional to the expression differential.,DNA chip,28,ppt课件,A microarray is a set of,第三节 真菌学诊断,标本,直接镜检,深部标本,染色标本,G,+,圆形,大小不均,有芽生孢子,分离培养,沙氏培养基,菌落观察,角质标本,不染色标本,10%,KOH,观察菌丝、孢子,类型 大小 颜色 皱摺,鉴定,动物实验,（,致病性真菌）,接种,观察发病情况,死亡后取标本进一步鉴定,皮肤超敏反应,快速诊断,血清学实验,核酸检测,真菌毒素检测,29,ppt课件,第三节 真菌学诊断标本直接镜检深部标本分离培养角质标本类,体癣,Tinea corporis,(ringworm):small lesions occurring anywhere on the body, not covered by hair and nail.Circular patches with advancing red, vesiculated border, pruritic.,Tinea corporis lesions on this patients,abdomen due to the fungus,Trichophyton rubrum.,www.hudlegekontoret.no,.,Skin scrapings taken from patients in 10% KOH, and show hyphae under microscope.,fungiqual-a.de,30,ppt课件,体癣Tinea corporis(ringworm):sma,新型隐球菌墨汁染色,Cryptococcus neoformans,India ink preparation demonstrating the large capsule surrounding budding yeast cells. The polysaccharide capsule can suppress T cell function and be considered a virulence factor,(magnification 1000).,31,ppt课件,新型隐球菌墨汁染色Cryptococcus neoforma,Microsporum canis,Culture of,M. canis,on mycobiotic agar showing a spreading whitish, cottony surface growth with a golden-yellow reverse pigment. Note non-pigmented strains may occur., 2006 The University of Adelaide,Candida albicans.,On Sabourauds dextrose agar colonies are white to cream colored, smooth, glabrous and yeast-like in appearance.,三种菌落:,酵母型菌落,类酵母型菌落,丝状菌落,32,ppt课件,Microsporum canis Culture of M,Reverse side of,T. rubrum,colony, showing deep red pigment,www.hudlegekontoret.no,Typical cigar shaped macroconidia of,红色毛癣菌,T. rubrum,granular type., 2006 The University of Adelaide,White, cottony surface,33,ppt课件,Reverse side of T. rubrum colo,芽管,Germ-tubes,produced in serum for 3 hrs at 37,o,C,厚膜孢子,Chlamydoconidia,on Difco chlamydospore agar. Trypan blue in the medium is absorbed strongly by these terminal vesicles. Numerous small blastoconidia and pseudohyphae are also present., 2006 The University of Adelaide,白假丝酵母菌,C. albicans,distinguish from other,Candida,species by:,34,ppt课件,芽管Germ-tubes厚膜孢子Chlamy,死疫苗,活疫苗,第四节 特异性预防与治疗,一、,人工主动免疫(,artificial active immunization)：,将疫苗(,vaccine),或类毒素接种于人体，使机体,主动,产生获得性免疫力的一种防治微生物感染的措施，主要用于预防。,35,ppt课件,死疫苗第四节 特异性预防与治疗一、人工主动免疫(artifi,活疫苗,死疫苗,制品特点,减毒或无毒的疫苗,死菌, 有免疫原性,接种次数,1次,2-3次,免疫类型,细胞和体液免疫,体液免疫,持续时间,长(1-5年),短(1年),毒力回升,可能,不可能,安全性,对免疫缺陷者有危险,安全性高,成本,高,低,36,ppt课件,类毒素: 外毒素经过0.3%-0.4%甲醛处理后, 失去毒性仍保持抗原性的生物制品.,37,ppt课件,类毒素: 外毒素经过0.3%-0.4%甲醛处理后, 失去毒性,抗毒素,抗菌血清,丙种球蛋白,二、 人工被动免疫,人工被动免疫(,artificial passtive immunization)：,注射含有特异性抗体的免疫血清或纯化免疫球蛋白抗体，或细胞因子等免疫制剂，使机体即刻获得特异性免疫的过程,，,因而作用及时。但这些免疫物质不是病人自己产生的，故维持时间短。主要用于治疗或紧急预防。,38,ppt课件,抗毒素二、 人工被动免疫人工被动免疫(artificial,两种人工免疫的比较,39,ppt课件,两种人工免疫的比较39ppt课件,Future vaccines,Synthetic peptides,that correspond to antigenic determinants on a viral protein.,Edible vaccines,. transgenic plants synthesizing antigens from pathogenic viruses.,From: www.niaid.nih.gov,40,ppt课件,Future vaccines From: www.niai,三、细菌感染的治疗,抗菌药物种类（人工或天然合成化学制剂）,主要作用机制:,影响细胞壁合成,影响细胞膜功能,影响细菌蛋白合成,影响细菌核酸合成,41,ppt课件,三、细菌感染的治疗抗菌药物种类（人工或天然合成化学制剂）41,革兰阳性菌,S.aureus,肽聚糖,(,peptidoglycan),: 聚糖骨架、四肽侧链、五肽交联桥.,转肽酶催化五肽交联桥的形成,青霉素抑制转肽酶, 从而抑制交联桥和细胞壁的形成,溶菌酶作用点,(,-1,4,glycosidic bond),N-,乙酰葡糖胺,N-,乙酰胞壁酸,42,ppt课件,革兰阳性菌S.aureus 肽聚糖(peptidoglyca,革兰阴性菌,E.coli,肽聚糖,(,peptidoglycan),: 聚糖骨架、四肽侧链,二氨基庚二酸,43,ppt课件,革兰阴性菌E.coli 肽聚糖(peptidoglycan),四、病毒感染的治疗,抗病毒化学制剂,核苷类药物,抑制病毒基因复制,反转录酶抑制剂,蛋白酶抑制剂,其他药物（金刚烷胺）,干扰素及干扰素诱生剂治疗,44,ppt课件,四、病毒感染的治疗抗病毒化学制剂44ppt课件,Activation of ACV (acycloguanosine) in herpes simplex virus-infected cells. ACV is converted to acycloguanosine monophosphate (acyclo GMP) by herpes-specific viral thymidine kinase, then to acycloguanosine triphosphate (acyclo GTP) by cellular kinases,阿昔洛韦:脱氧鸟嘌呤,dG,类似物,45,ppt课件,Activation of ACV (acycloguano,From: Dr. R.Hunt,46,ppt课件,From: Dr. R.Hunt46ppt课件,五、真菌的治疗,各种癣症,外用抗真菌药,深部真菌病,除去诱因，提高免疫力，用抗真菌药物,47,ppt课件,五、真菌的治疗各种癣症 外用抗真菌药47ppt课件,