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Laboratory Diagnosis

Laboratory Diagnosis. Category of Sample. Blood, Urine, Stool, nasal washing, nasal swab , throat swab, saliva , sputum, rectal swab, vesicle fluid( scraping or swab), tissue ,brain biopsy, cerebrospinal fluid, et al. Laboratory Diagnosis. Microscopy Identification

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Laboratory Diagnosis

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  1. Laboratory Diagnosis

  2. Category of Sample • Blood, Urine, Stool, nasal washing, nasal swab , throat swab, saliva , sputum, rectal swab, vesicle fluid( scraping or swab), tissue ,brain biopsy, cerebrospinal fluid, et al.

  3. Laboratory Diagnosis • Microscopy Identification • Virus isolation and identification • Detection of viral proteins( antigens and enzymes)] • Detection of viral genetic material • Serologic procedures

  4. Microscopy Identification • Light microscopy • Fluorescent microscopy • Electron microscopy

  5. Light microscopy • Characteristic CPE • Inclusion Bodies

  6. Cell death Cell rounding Degeneration Aggregation Loss of attachments to substrate • Characteristic histological changes:inclusion bodies in the nucleus or cytoplasm, margination of chromatin • Syncytia:multinucleated giant cells caused by virus-induced cell-cell fusion

  7. Fluorescent microscopy • Fluorescent-antibody staining

  8. Electron microscopy • Direct detection : Human rotavirus; HAV; HBV; Smallpox virus; Herpes virus. • Immune Electron microscopy: Human rotavirus; HAV;

  9. Laboratory Diagnosis • Microscopy Identification • Virus isolation and identification • Detection of viral proteins( antigens and enzymes)] • Detection of viral genetic material • Serologic procedures

  10. Viral isolation and Identification • Viral Growth and Cell culture • Viral Detection • Viral Identification • Interpretation of culture results

  11. Systems for the Propagation of Viruses • People • Animals: cows, chickens, mice,rats, suckling mice • Embryonated eggs • Organ and tissue culture Organ culture Primary tissue culture Cell lines: diploid Tumor or （immortalized ）cell line

  12. Viral detection • CPE • Hemadsorption • Interfere • Metabolize of cell

  13. TCID50(Tissue culture infective dose) • TCID50 is defined as that dilution of virus which will cause CPE in 50% of a given batch of cell culture • TCID50= log10of highest dilution giving 100%CPE +1/2 – (total number of test units showing CPE)/ (number of test units per dilution)

  14. Viral identification • Complement fixation： • Hemagglutination inhibition • Neutralization • Immunofluorescence ( direct or indirect) • Latex agglutination • In situ EIA • ELISA • RIA(radioimmuno

  15. Laboratory Diagnosis • Microscopy Identification • Virus isolation and identification • Detection of viral proteins( antigens and enzymes) • Detection of viral genetic material • Serologic procedures

  16. Detection of viral proteins( antigens and enzymes) • Antigen detection ( ELISA, RIA, Western blot) • Hemagglutination and hemadsorption • Enzyme activities( reverse transcriptase) • Protein patterns( electrophoresis )

  17. Laboratory Diagnosis • Microscopy Identification • Virus isolation and identification • Detection of viral proteins( antigens and enzymes)] • Detection of viral genetic material • Serologic procedures

  18. Detection of viral genetic material • PCR ( Polymerase chain reaction) • RT-PCR (Reverse transcriptase polymerase chain reaction) • Southern（DNA), Northern(RNA), and dot blots • DNA genome hybridization in situ(cytochemistry) • Electrophoretic mobilities of RNA for segmented RNA viruses( Electrophoresis) • Restriction endonuclease cleavage patterns

  19. Laboratory Diagnosis • Microscopy Identification • Virus isolation and identification • Detection of viral proteins( antigens and enzymes)] • Detection of viral genetic material • Serologic procedures

  20. Serologic procedures • If the antibody titer in the convalesent-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. • In certain viral diseases, the presence of IgM antibody is used to diagnose current infection • Other nonspecific serologic tests are available

  21. Serologic procedures • Complement fixation： • Hemagglutination inhibition • Neutralization • Immunofluorescence ( direct or indirect) • Latex agglutination • In situ EIA • ELISA • RIA

  22. Viruses Diagnosed by Serology • Epstein-Barr virus • Rubella virus • Hepatitis A, B, C, D, and E viruses • HIV • Human T-cell Leukemia virus • Arboviruses ( Encephalitis viruses)

  23. Prevention • Successes of the Past • Possibilities for the Future

  24. Active immunization Vaccines

  25. Overview of Active immunization • Active immunization - administration of antigen resulting in production of a specific immune response with immunologic memory. Response may be cellular or humoral or both. • Natural immunity - to diseases you have caught and successfully fought • Artificial immunity –Vaccination(vaccines)

  26. Attributes of a good vaccine • Ability to elicit the appropriate immune response for the particular pathogen • Long term protection ideally life-long • Safety vaccine itself should not cause disease • Stable retain immunogenicity, despite adverse storage conditions prior to administration • In-expensive

  27. LIVE VACCINES • Live attenuated organism • Heterologous vaccines • Live recombinant vaccines • Attributes – live vaccines

  28. Live attenuated organism • Organisms whose virulence has been artificially reduced by in vitro Culture under adverse conditions, such as reduced temperature.

  29. Heterologous vaccines • Closely related organism of lesser virulence, which shares many antigens with the virulent organism. The vaccine strain replication in the host and induces an immune response that cross reacts with antigens of the virulent organism. • Vaccinia virus /cowpox virus--- Variola virus

  30. Live recombinant • Vector • bovine vaccine • BCG

  31. Advantages of Attenuated Vaccines 2-1 • Both cell mediated immunity and antibody response • Activates all phases of immune system. Can get humoral IgG and local IgA • Raises immune response to all protective antigens. Inactivation may alter antigenicity. • More durable immunity; more cross-reactive • Immunity is long lived • Single dose

  32. Advantages of Attenuated Vaccines 2-2 • Low cost • Quick immunity in majority of vaccinees • In case of polio and adeno vaccines, easy administration • Easy transport in field • Can lead to elimination of wild type virus from the community

  33. Disadvantages of Live Attenuated Vaccine • Mutation; reversion to virulence (often frequent) • Spread to contacts of vaccinee who have not consented to be vaccinated (could also be an advantage in communities where vaccination is not 100%) • Spread vaccine not standardized--may be back-mutated • Poor "take" in tropics • Problem in immunodeficiency disease (may spread to these patients)

  34. Killed vaccines • The organism is propagated in bulk, in vitro, and inactivated with either beta-propiolactone or formaldehyde. These vaccines are not infectious and are therefore relatively safe. However, they are usually of lower immunogenicity and multiple doses may be needed to induce immunity. In addition, they are usually expensive to prepare.

  35. Killed vaccines • Inactivated organism: rabies virus; epidmic type B encephalitis virus. • Subunit Vaccines: Influenza virus( HA and NA) • Recombinant proteins: HBV

  36. Advantages of inactivated vaccines • Gives sufficient humoral immunity if boosters given • No mutation or reversion • Can be used with immuno-deficient patients • These vaccines tend to be able to withstand more adverse storage conditions,Sometimes better in tropics

  37. Disadvantages of inactivated vaccines • Many vaccinees do not raise immunity • poor, only antibody, no cell immediated immune response • response is short-lived and multiple doses are needed • No local immunity (important) • Inactivated, therefore can not replicate in the host and cause disease • Failure in inactivation and immunization with virulent virus • Expense: Expensive to prepare

  38. New Methods • Selection of attenuated virus strain • Varicella • Hepatitis A • Use monoclonal antibodies to select for virus with altered surface receptor • Rabies • Reo • Use mutagen and grow virus at 32 degrees. Selects for temperature-sensitive virus. Grows in upper respiratory tract but not lower • ‘flu (new vaccine) • respiratory syncytial virus

  39. New Methods Passage progressively at cold temperatures TS mutant in internal proteins Can be re-assorted to so that coat is the strain that is this years flu strain

  40. X PB2 PB2 PB1 PB1 PA PA HA HA NA NA NP NP M M NS NS PB2 PB1 New Virulent Antigenic Variant Strain Attenuated Donor Master Strain PA HA NA NP Attenuated Vaccine Strain: Coat of Virulent strain with Virulence Characteristics of Attenuated Strain M NS

  41. New Methods • Deletion mutants • Suppression unlikely (but caution in HIV) • Viable but growth restrictions • Problems • Oncogenicity in some cases (adeno, retro)

  42. Recombinant DNA • Single gene (subunit) S-antigen mRNA Hepatitis B vaccine raised in yeast cDNA Express plasmid S-antigen mRNA protein New Methods

  43. Single gene (subunit) - problems • Surface glycoprotein poorly soluble - deletion? • Poorly immunogenic • Post-translational modifications • Poor CTL response

  44. Single gene (subunit) in expression vector • Vaccinate with live virus • Canary Pox • Infects human cells but does not replicate • Better presentation • CTL response • Vaccinia • Attenuated Polio • Being developed for anti-HIV vaccine

  45. New Methods • Chemically synthesized peptide • malaria • poorly immunogenic

  46. antibody New methods Anti-idiotype vaccine Virus epitope Antibody with epitope binding site

  47. Anti-idiotypeantibody antibody Anti-idiotype vaccine cont Make antibody against antibody idiotype Anti-idiotype antibody mimics the epitope

  48. Anti-idiotype antibody cont 2 Anti-idiotypeantibody Anti-anti-idiotypeantibody Anti-anti-idiotypeantibody Anti-anti-idiotypeantibody Use anti-idiotype antibody as injectable vaccine Use as vaccine Binds and neutralizes virus Antibody to anti-idiotype antibody

  49. New Methods • New “Jennerian Vaccines” • Live vaccines derived from animal strains of similar viruses • Naturally attenuated for humans • Rotavirus: Monkey Rota • 80% effective in some human populations • Ineffective in others • Due to differences in circulating viral serotypes

  50. New Methods • New Jennerian Vaccines • Bovine parainfluenza Type 3 • Bovine virus is: • Infectious to humans • Immunogenic (61% of children get good response) • Poorly transmissable • Phenotypicaly stable

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