感染性疾病实验诊断

1、关于感染性疾病的实验诊断第一张，PPT共一百三十二页，创作于2022年6月前 言实验诊断学(laboratory diagnosis) 是诊断学的一部分,它是基础医学向临床医学过渡的桥梁课程之一。实验诊断学的任务是将临床检验所提供的检验信息，通过医师的分析和思维，科学地应用于临床诊断、鉴别诊断、观察病情、判断预后、预防疾病的一门学科，特别强调在循证实验医学的原则下推行“实验的优化组合应用”和“在实验监测下作个体化治疗的应用”，实验诊断学也可称为临床检验诊断学。因此，它是医学生的必修课，必须加以重视。第二张，PPT共一百三十二页，创作于2022年6月 感染性疾病的实验诊断第三张，PPT共一百三十

2、二页，创作于2022年6月主要学习内容及要求掌握感染性疾病的常用实验室检测项目及其临床意义和评价不同病原体感染性疾病实验室检测与不同系统病原体感染性疾病实验室检测特点熟悉引起血流感染、中枢神经系统感染、呼吸系统感染、 消化系统感染、 泌尿系统感染、皮肤及软组织感染和先天及新生儿感染常见病原体与常见疾病及病原学检查、常见医院感染病原体及特点、医院感染的微生物学检测及医院感染暴发流行的识别和处理了解当今感染病特点、感染类型第四张，PPT共一百三十二页，创作于2022年6月Specimen Collection Successful laboratory diagnosis of a microbi

3、al infection depends on many factors beginning with a well-collected sample. Proper specimen selection, collection, and transport are all essential to ensure that a specimen is representative of the disease process and minimally contaminated with microorganisms present in adjacent tissues. 第五张，PPT共一

4、百三十二页，创作于2022年6月Site and TimingCollect the sample from the correct anatomic site . eg. a superficial sample of a lesion is not useful in identifying the cause of a deep wound infection. The timing of sample collection is also important. eg, when submitting a specimen for bacterial culture, samples s

5、hould be collected before the administration of antibiotics 第六张，PPT共一百三十二页，创作于2022年6月Collection TechniquesSufficient volume Sterile technique and equipment. After collection, the specimen must be placed in an appropriately labeled leak-proof container. 第七张，PPT共一百三十二页，创作于2022年6月Transport of Specimens

6、Rapid, optimally in less than 2 hours. For delays in transport, most specimens should be refrigerated.Exceptions: blood, cerebrospinal fluid (CSF), and specimens to be examined for anaerobes, fastidious organisms such as Neisseria gonorrboeae and Bordetella pertussis, and Trichomonas vaginalis, all

7、of which should be maintained at room temperature. 第八张，PPT共一百三十二页，创作于2022年6月难点/重点解读（一）细菌感染性疾病常用实验室检测项目的组合（二）病毒感染性疾病常用实验室检测项目的组合（三）真菌感染性疾病常用实验室检测项目的组合（四）寄生虫感染性疾病常用实验室检测项目的组合第九张，PPT共一百三十二页，创作于2022年6月细菌感染性疾病 常用实验室检测项目的组合 1. 感染病的实验诊断方法虽按基本原则进行，但细感染性疾病具下述特点： 在检查项目中，尤其以细菌分离培养鉴定为主。在无菌标本中检查到细菌，则此细菌为感染的病原；存在

8、正常菌群的标本中检查到细菌，应区分是正常寄居的菌群或是致病的细菌，常需要作细菌定量计数，根据细菌数判断是否为感染的病原菌。 第十张，PPT共一百三十二页，创作于2022年6月细菌感染性疾病 常用实验室检测项目的组合2. 根据目的不同，检测项目的选择是： （1）诊断疾病的病原学，一般情况下只需鉴定到细菌的 种，必要时再进一步鉴定； （2）提供治疗方案，可以进行临床标本的直接药物敏感 试验，还可根据分离出病原菌的种类直接提供选择 抗生素的范围和种类； （3) 研究流行病学，必须对病原菌做进一步鉴定，往往 鉴定到型（血清型或基因型）的水平； (4) 要了解这类细菌的致病性，必须进行细致鉴定，包 括毒

9、素、侵袭性酶类的检测。 第十一张，PPT共一百三十二页，创作于2022年6月（一）细菌感染性疾病常用实验室检测项目的组合 3.根据标本来源，通常构成不同组合的检验方法： （1）无菌性标本（血液、脑脊液、体腔渗出液），采用显微镜检查和直接药敏试验可作为病原学诊断和提供临床治疗用药指导； （2）脓血便，采用革兰染色无诊断意义，需用标记抗体染色镜检获初步报告，再需经选择培养基分离培养，挑取可疑菌落作玻片血清凝集，阳性者可初步鉴定，再用配套生化反应可作出最后报告； （3）尿液标本，显微镜检查可初步估计细菌菌量，作出初步诊断，再经分离培养鉴定后和菌落定量计数可作出病原学诊断；痰标本，采用显微镜检查，判定

10、是否为合格标本，若为合格标本作分区划线接种，检出致病性菌落，再进行鉴定和药敏。 第十二张，PPT共一百三十二页，创作于2022年6月细菌感染性疾病常用实验室检测项目的组合4.结果报告可由实验室人员可分别出具： 先后和程度不同报告各检测项目（显微镜检查、 分离培养、血清学试验、生化鉴定和PCR等核酸 检测）结果，为临床提供信息。5.为保证细菌感染正确诊断，必须遵循下述细菌感染诊断原则：要求医师和检验师的密切配合，不应停留在化验单往来；重视检验标本的采集和送检；重视检验过程的阶段性和连续性；检验程序需有针对性和综合性。第十三张，PPT共一百三十二页，创作于2022年6月Identifying Ba

11、cteria that Cause DiseasePrinciples of Diagnostic Medical MicrobiologyIntroductionDiagnostic medical microbiology is concerned with the etiologic diagnosis of infection. Laboratory procedures used in the diagnosis of infectious disease in humans include the following:第十四张，PPT共一百三十二页，创作于2022年6月Identi

12、fying Bacteria that Cause DiseasePrinciples of Diagnostic Medical Microbiology(1) Morphologic identification of the agent in stains of specimens or sections of tissues (light and electron microscopy).(2) Culture isolation and identification of the agent.(3) Detection of antigen from the agent by imm

13、unologic assay (latex agglutination, EIA, etc) or by fluorescein-labeled (or peroxidase-labeled) antibody stains.第十五张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseasePrinciples of Diagnostic Medical Microbiology(4) DNA-DNA or DNA-RNA hybridization to detect pathogen-specific genes in pat

14、ients specimens.(5) Detection and amplification of organism nucleic acid in patients specimens.(6) Demonstration of meaningful antibody or cell-mediated immune responses to an infectious agent.第十六张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseasePrinciples of Diagnostic Medical Microbiol

15、ogyPhysicians who deal with infectious processes must know when and how to take specimens, what laboratory examinations to request, and how to interpret the results. Clinical information is much more important for diagnostic microbiology than it is for clinical chemistry or hematology. The clinician

16、 must make a tentative diagnosis rather than wait until laboratory results are available. 第十七张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseasePrinciples of Diagnostic Medical MicrobiologyCommunication between Physician & Laboratory As the laboratory staff begins to obtain results, they

17、inform the physician, who can then reevaluate the diagnosis and clinical course of the patient and perhaps make changes in the therapeutic program. This feedback information from the laboratory consists of preliminary reports of the results of individual steps in the isolation and identification of

18、the causative agent.第十八张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause Disease Diagnosis of Bacterial InfectionsSpecimensLaboratory examination usually includes microscopic study of fresh unstained and stained materials and preparation of cultures with conditions suitable for growth of a wide

19、 variety of microorganisms, including the type of organism most likely to be causative based on clinical evidence. 第十九张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of Bacterial InfectionsSpecimensIf a microorganism is isolated, complete identification may then be pursued. I

20、solated microorganisms may be tested for susceptibility to antimicrobial drugs. When significant pathogens are isolated before treatment, follow-up laboratory examinations during and after treatment may be appropriate.第二十张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of Bact

21、erial Infections SpecimensSpecimensA properly collected specimen is the single most important step in the diagnosis of an infection, because the results of diagnostic tests for infectious diseases depend upon the selection, timing, and method of collection of specimens. 第二十一张，PPT共一百三十二页，创作于2022年6月Id

22、entifying Bacteria that Cause DiseaseDiagnosis of Bacterial Infections SpecimensSpecimensRecovery of bacteria is most significant if the agent is isolated from a site normally devoid of microorganisms (a normally sterile area). Any type of microorganism cultured from blood, cerebrospinal fluid, join

23、t fluid, or the pleural cavity is a significant diagnostic finding. Conversely, many parts of the body have a normal microbial flora( respiratory, gastrointestinal, or genitourinary tracts; from wounds; or from the skin) must be considered in the context of the normal flora of each particular site.

24、第二十二张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of Bacterial Infections SpecimensSpecimensA few general rules apply to all specimens:(1) The quantity of material must be adequate.(2) The sample should be representative of the infectious process (eg, sputum, not saliva; pu

25、s from the underlying lesion, not from its sinus tract; a swab from the depth of the wound, not from its surface). (3) Contamination of the specimen must be avoided by using only sterile equipment and aseptic precautions.第二十三张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of

26、Bacterial Infections Specimens(4) The specimen must be taken to the laboratory and examined promptly. Special transport media may be helpful.(5) Meaningful specimens to diagnose bacterial and fungal infections must be secured before antimicrobial drugs are administered. If antimicrobial drugs are gi

27、ven before specimens are taken for microbiologic study, drug therapy may have to be stopped and repeat specimens obtained several days later.第二十四张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of Bacterial Infections Microscopy & StainsGram staining is a very useful procedure

28、 in diagnostic microbiology. Most specimens submitted when bacterial infection is suspected should be smeared on glass slides, Gram-stained, and examined microscopically. 第二十五张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of Bacterial Infections Microscopy & StainsThe appear

29、ance of bacteria on Gram-stained smears does not permit identification of species. Reports of gram-positive cocci in chains are suggestive of, but not definitive for, streptococcal species; gram-positive cocci in clusters suggest a staphylococcal species. 第二十六张，PPT共一百三十二页，创作于2022年6月Identifying Bacte

30、ria that Cause DiseaseDiagnosis of Bacterial Infections Microscopy & StainsSpecimens submitted for examination for mycobacteria should be stained for acid-fast organisms, using either Ziehl-Neelsen stain or Kinyoun stain. Immunofluorescent antibody (IF) staining is useful in the identification of ma

31、ny microorganisms 第二十七张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of Bacterial InfectionsCulture SystemsFor diagnostic bacteriology, it is necessary to use several types of media for routine culture, particularly when the possible organisms include aerobic, facultatively

32、anaerobic, and obligately anaerobic bacteria. 第二十八张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of Bacterial InfectionsCulture Systems culture media used to diagnose : blood agar, Chocolate agar, selective medium for enteric gram-negative rods (either MacConkey agar or eosi

33、n-methylene blue EMB agar,brucella agar with hemin and vitamin K, Many other specialized media are used in diagnostic bacteriology; choices depend on the clinical diagnosis and the organism under consideration. 第二十九张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of Bacterial

34、Infections Antigen DetectionImmunologic systems designed to detect antigens of microorganisms can be used in the diagnosis of specific infections. IF tests (direct and indirect fluorescent antibody tests) are one form of antigen detection and are discussed in separate sections in this chapter on the

35、 diagnosis of bacterial, chlamydial, and viral infections and in the chapters on the specific microorganisms.第三十张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of Bacterial Infections Antigen DetectionEnzyme immunoassays (EIA), including enzyme-linked immunosorbent assays (EL

36、ISA), and agglutination tests are used to detect antigens of infectious agents present in clinical specimens. The principles of these tests are reviewed briefly here. 第三十一张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseDiagnosis of Bacterial Infections Antigen DetectionLatex agglutinat

37、ion tests are primarily directed at the detection of carbohydrate antigens of encapsulated microorganisms. Another form of EIA, to detect antibody, is immunoblotting (Western blot), whereby defined antigens are placed on strips of nitrocellulose paper. 第三十二张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria

38、 that Cause DiseaseBacteria Using 16S rRNAThe 16S rRNA of each species of bacteria has stable (conserved) portions of the sequence. Many copies are present in each organism. Labeled probes specific for the 16S rRNA of a species are added, and the amount of label on the double-stranded hybrid is meas

39、ured. 第三十三张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseBacteria Using 16S rRNAThis technique is widely used for the rapid identification of many organisms. Examples include the most common and important Mycobacterium species, Coccidioides immitis, Histoplasma capsulatum, and others.

40、第三十四张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseMolecular DiagnosticsThe principle behind early molecular assays is the hybridization of a characterized nucleic acid probe to a specific nucleic acid sequence in a test specimen followed by detection of the paired hybrid. 第三十五张，PPT共一

41、百三十二页，创作于2022年6月Identifying Bacteria that Cause DiseaseMolecular DiagnosticsTarget Amplification Systems transcription-mediated amplification (TMA)the nucleic acid sequence-based amplification (NASBA) Strand displacement assays (SDA) Probe Amplification Systems-The ligase chain reaction (LCR ) Signa

42、l Amplification Techniques-branched DNA (bDNA) 第三十六张，PPT共一百三十二页，创作于2022年6月Identifying Bacteria that Cause Disease第三十七张，PPT共一百三十二页，创作于2022年6月病毒感染性疾病 常用实验室检测项目的组合 1.感染病的实验诊断方法虽按基本原则进行，但 病毒感染性疾病具下述特点： 作分离的标本和细菌标本不一样，必须是经 滤过除菌的液体标本。大多数病毒对热敏感 ， 因此标本需迅速冷藏和运送。病毒分离培养后, 根据细胞病变特征确定何种病毒，再此基础 上，对已分离的病毒和已知参考血清作中

43、和试 验、补体结合试验。 第三十八张，PPT共一百三十二页，创作于2022年6月病毒感染性疾病 常用实验室检测项目的组合2.病毒血清学检查 是病毒感染诊断的常规实验室方法，用免疫标记技术检测标本组织细胞内的病毒抗原和胞外游离病毒抗原是一种快速的早期诊断。显微镜检查也是病毒实验诊断不可忽视的手段，光学显微镜检查感染组织或脱落细胞中特征性病毒包涵体、电镜检查病毒颗粒是早期诊断的方法之一。第三十九张，PPT共一百三十二页，创作于2022年6月病毒感染性疾病 常用实验室检测项目的组合3.核酸检测 病毒分离鉴定和血清学诊断一般需较长时间才能判断结果，近年来发展的利用核酸杂交技术和PCR技术检测标本中病毒

44、核酸，是一种快速的早期诊断。对那些可能新出现的病毒，只有用分离方法取得最好诊断结果。第四十张，PPT共一百三十二页，创作于2022年6月病毒感染性疾病 常用实验室检测项目的组合 4.以循证检验为依据选择实验项目 病毒感染实验诊断方法诸多，且各具不同的敏感度和特 异度。有关的医学专业组织可充分利用已有的循证诊断 试验的文献，制定科学的、严格的、用于临床疾病诊断 的指南。指南根据现有的最佳证据或专家的一致意见， 提供诊断建议，将实验室诊断试验项目分别按证据的等 级水平进行标记。临床实验室人员、特别是临床医师可 依据指南对同一组织器官、不同病毒感染或不同组织器 官、同一病毒感染选择最佳。第四十一张，

45、PPT共一百三十二页，创作于2022年6月approaches to the diagnosis of viral infectionThere are two major approaches to the diagnosis of viral infection: virologic and serologic. The virologic approach includes (1) isolation of infectious virus in cell culture or in laboratory animals, (2)detection of viral antigenby

46、immunologic methods such as immunofluorescence assay (IFA) or enzyme immunoassay (EIA), (3) identification of viral particlesby electron microscopy (EM), and (4) detection of viral nucleic acid. 第四十二张，PPT共一百三十二页，创作于2022年6月approaches to the diagnosis of viral infectionThe serologic approach includes

47、(1)demonstration of a fourfold or greater rise in antiviral antibody using acute and convalescent sera and a variety of methods that measure predominantly immunoglobulin (Ig) antibody and (2)demonstration of virus-specific IgM antibody in a single late acute or recovery phase serum.第四十三张，PPT共一百三十二页，

48、创作于2022年6月approaches to the diagnosis of viral infectionAs is true for any diagnostic test, the timing, quality, and handling of the specimen are critical for an accurate diagnosis. For the virologic approach to diagnosis, it is important to obtain specimens during the acute infection from the site

49、of disease. 第四十四张，PPT共一百三十二页，创作于2022年6月approaches to the diagnosis of viral infectionOptimal Specimens The optimal specimens vary depending on the site of disease： 1、Lesions 2、Pediatric Respiratory Illness 3、Adult Respiratory Illness4、Acute Infection第四十五张，PPT共一百三十二页，创作于2022年6月 approaches to the diag

50、nosis of viral infection- Viral IsolationIsolation of infectious virus in tissue cell cultures remains one of the most commonly used methods to detect viruses. The major viruses detected by isolation in cell culture include HSV,CMV,RSV, influenza A and B ,the parainfluenza viruses, the adenoviruses,

51、 the enteroviruses (Coxsackie A and B,echo, polio), and VZV. 第四十六张，PPT共一百三十二页，创作于2022年6月 approaches to the diagnosis of viral infection- Viral IsolationSeveral Different Types of Cells Because not all culturable viruses will grow in a single cell line, several different types of cells are required:

52、(1)During the winter season, the cells that may be used to isolate respiratory viruses include primary rhesus monkey kidney. Madin-Darby canine kidney, Hep-2(a human epithelial carcinoma cell line,) buffalo green monkey kidney continuous cell line, and a human fibroblast cell. 第四十七张，PPT共一百三十二页，创作于20

53、22年6月 approaches to the diagnosis of viral infection- Viral IsolationSeveral Different Types of Cells (2) For the summer/fall enterovious season,these are different from the cells used: primary rhesus monkey kidney, Hep-2, buffalo green monkey kidney, RD(a human rhabdomyosarcoma cell line),and a hum

54、an fibroblast cell. 第四十八张，PPT共一百三十二页，创作于2022年6月approaches to the diagnosis of viral infection -Viral Isolation(3)Cytopathic Effect (CPE) 1.Many viruses will demonstrate a cytopathic effect (CPE) within a few days, whereas some may take as long as a week or two . Some viruses will do not cause typica

55、l CPE and are identified by the adsorption of red blood cells to the surface of virus-infected cells in the culture (e.g hemadsorption for the influenza and parainfluenza viruses). For some viruses, such as several of the Coxsackie A viruses and Colorado tick fever virus mouse inoculation is require

56、d. 第四十九张，PPT共一百三十二页，创作于2022年6月Cytopathic Effect (CPE)第五十张，PPT共一百三十二页，创作于2022年6月 hemadsorption for the influenza and parainfluenza viruses第五十一张，PPT共一百三十二页，创作于2022年6月mouse inoculation 第五十二张，PPT共一百三十二页，创作于2022年6月approaches to the diagnosis of viral infection -Viral IsolationCytopathic Effect (CPE) 2.Wi

57、th cell culture systems, presumptive identification of particular virus or virus group(e.g. HSV or RSV or an enterovirus)can be performed within a few or several days based on the CPE: characteristics, time of onset ,and cell type in which it occurs. This presumptive identification can be greatly fa

58、cilitated if the test requisition slip contains the important information requested: source of the specimen (e.g. swab of a genital ulcer or a conjunctival swab rather than an unidentified swab source ) and clinical diagnosis(e.g. genital herpes, influenza, aseptic meningitis).第五十三张，PPT共一百三十二页，创作于20

59、22年6月approaches to the diagnosis of viral infection -Viral IsolationConfirmation of a Specific Virus Confirmation of a specific virus serotype or of a virus (e.g.HSV-2, influenza A H3N2, echovirus11)requires use of immunologic methods with antibody of known specificity. In recent years, fluorescence

60、 or peroxidase-conjugated monoclonal antibodies(MABs)have become available commercially to detect viral antigen in cell cultures, e.g. HSV, CMV, VZV, RSV, influenza A and B, the parainfluenza viruses, and the adenoviruses. 第五十四张，PPT共一百三十二页，创作于2022年6月approaches to the diagnosis of viral infection -Vi