Chapter 12 principles of diagnosis and prevention of pathogen infection

1. Chapter 12 principles of diagnosis and prevention of pathogen infection

2. Bacteriological diagnosis※ • Virology diagnosis※ • Mycology diagnosis • Specific prophylaxis and therapy

3. section 1 bacterial diagnosis bacteriological diagnosis: ◙check pathogen and their components（Ag ,products ,nucleic acid） detect Ag with known Ab— serological identification ◙detect Ab in serum: detect unknown Ab with known Ag— serodiagnosis

4. Steps precede the laboratory work • Choose the appropriate specimen • Obtain the specimen, avoid contaminated • Transport the specimen promptly or store it correctly • Provide essential information

5. （二）detection of pathogenic bacteria 1、morphology and structure 2、isolating culture 3、detection of Ag 4、other methods

6. 1、morphosis detection ● procedure： smear fix stain observe under microscope （shape,size,arrangement, staining, special structure） identify bacteria

7. ●major technique for detection of pathogenic bacteria （1）microscope A.light microscope resolution： 0.25μm magnification：×1000

8. B. electronmicroscope resolution：1nm magnification： ×100000 --cannot observe viable organism

9. C. Other microscope •darkfield microscope •contrast phase microscope •fluorescence microscope •confocal microscope

10. （2）staining technique simple staining：only use one dye , observe morphology ,arrangement compound staining：use 2 kinds dyes or more, to identify bacteria

11. Gram staining：positive（violet）、 negative（red） Acid-fast staining：positive（red）、 negative （blue） Special staining：for bacterial capsule ,spore ,flagella ,and so on.

12. 2、isolating culture and identify General procedure： cultural feature: nutrition, growing condition, Colony feature , hemolysis pure Plain agar Blood agar Selective medium specimen inoculate stain：initial identify (morphology) Biochemical reaction

13. Serological identification：species, groups detect endotoxin or exotoxin animal test：rabbit, guinea pig Virulence identification Slip method：determine the nature Test tube method：quantitation（detect MBC、MIC） Susceptibility test

14. 3、detect Ag ——serological identification highly specificity sensitivity

15. 4、other detection： • Detect metabolic product： • PCR: detect DNA caution: avoid contamination (faulse positive) • DNA chips technique

16. 2、serological diagnosis -----detect Ab with known Ag ------specimens：serum ------paired serum (acute； convalescent serum)

17. Compared acute serum and convalescent serum quantitatively • A 4-fold or greater increase in Ab titer supports a diagnosis of recent infection • Serum containing a high titer of Abs of the IgM subclass would suggest a current infection

18. Agglutination test： Widal test---diagnose typhus and paratyphoid Well-Felix---diagnose rickettsiosis 􀁨 Neutralization test： anti“O” test---diagnose rheumatic disease ●commonly used methods：

19. Section 2 virology diagnosis Electron microscope Light microscope : inclusion body isolation Virusparticle V Ag V nucleic acid Viral enzyme：retroviridase Detectvirus Viralinfection DetectAb

20. Specimen Selection, Collection, and Processing • Choose the appropriate specimen • Aseptic operation，add antibiotics to specimen • Take it in acute phase • Low temperature conservation，transport it promptly，preserve in 50％ glycerine • Serologial diagnose: paired serum

21. 二、viral isolation and identifying positive negative identifying Animal inoculation Embryonated egg Cell culture asepsis specimen Blindpassage 2－3generation negative Virus negative

22. （一）viral isolation 1、animal inoculation: chimpanzee,monkey,rabbit,rat

23. 2、embryonated egg inoculation:

24. 3. Cell culture（commonly used） Primary and secondary cell culture general procedure： brokenprotease tissuetissuepiecesporadicsinglecell cell monolayer (primary cell) Serial subcultivation Cell monolayer (secondary cell culture)

25. ●features of virus cell culture A. sensitive to many kinds of viruses B. cost high C. carry latent virus

26. (2)Diploidcellculture usedto isolate virus and obtain vaccine

27. (3) secondary cell culture . sensitive to multiple viruses . high reproductive capacity ,long generation time . have danger of carcinogenesis，cannot use to produce vaccine

28. 1. Cytopathic effect, CPE) (二)indexfor reproduction of virus (1)Virusinfected cell （cell rounding,gather, necrosis）

29. CPE （2）cell fusion after virus infection, Multinucleated giant cell formation

30. CPE （3）viral inclusion in cytoplasm or nucleus of infected cell

31. 2. hemadsorption, HAd hemagglutinin（HA）:on membrane of virus infected cell. Can adsorb vertebrate RBC

32. 3. Interference 4. Cell metabolism change： Virus reproduction，pH of culture change

33. (三) viral quantitative assay Hemagglutination test：total amount Plaque bacteriophage determination ID50 or TCID50

34. Section 3 mycology diagnosis (一) collection of specimens Superficial infected fungus：surface layer skin lesion tissue deep infected fungus：body fluid ,secretion , excretion (二)detection and identify 1、direct observation with microscope：spore and hypha

35. 2、culture： specimen sabouraud medium macroculture microculture colony biochemical reaction observe or hypha molecular biology technique spore

36. 3.animal test 4.skin test: hypersensitivy （三）mycology rapid diagnosis serological test :detect Ag or Ab detect nucleic acid of fungus detect mycotoxin

37. questions 1、definition serological identification and diagnosis) Cytopathic effect, CPE hemadsorption, HAd interference 2、principle of Widal’s test 3、 principle of Gram’s staining

38. Section 4 artificial active immunity

39. Artificial active immunity stimulate the body’s immune mechanisms through administration of a vaccine or toxoid

40. vaccines • Capsular polysaccharides • Inactivated protein exotoxins (toxoids) • killed bacteria • Live attenuated B. • Subcellular fragments • Genes for Ags in some vectors • DNA

41. A. Live (attenuated) vaccines • Consist of organisms attenuated by growth in unfavourable conditions • The genes of organisms mutate • Mutants lost virulence but retain antigenicity are repeatedly selected • BCG

42. Conditional-lethal mutations The mutation is expressed only under certain conditions may be useful in vaccines

43. temperature-sensitive conditional-lethal mutation the organisms can replicate at a relatively low, permissive temperature, but can’t grow at a higher, restrictive temperature

44. A. Live (attenuated) vaccines • Can regain virulence by backmutation • Often induce stronger and better localized immunity • Induce more appropriate response • Do not often require adjuvants or “booster” injection

45. B. Inactivated (dead) vaccines • Killed organisms or B. products • Induce weak and/or inappropriate response • Immune memory may be variable or poor • Usually safe

46. toxoids • Formaldehyde-treated exotoxin • Induce antitoxin Abs • No immunity against the B. themselves

47. artificial passive immunization

48. Artificial passive immunization • Administer either preformed immunoreactive serum or cells • IgG is predominant • Obtained from human or animal donors who have recovered from an infectious disease or been immunized

49. Artificial passive immunization • Provide immediate protection • Be useful for individuals • Cannot form Abs • Before Ab production • animal sera have the risk of allergic reactions