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Laboratory Diagnosis

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Title: Laboratory Diagnosis


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 changesinclusion
    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 log10 of highest dilution giving 100CPE
    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
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41
New Methods
  • Deletion mutants
  • Suppression unlikely (but caution in HIV)
  • Viable but growth restrictions
  • Problems
  • Oncogenicity in some cases (adeno, retro)

42
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
New methods
Anti-idiotype vaccine
Virus
epitope
Antibody with epitope binding site
47
Anti-idiotype vaccine cont
Make antibody against antibody idiotype
Anti-idiotype antibody mimics the epitope
48
Anti-idiotype antibody cont 2
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

51
New Methods
52
Vaccines
  • 1796 Jenner wild type animal-adapted virus
  • 1800s Pasteur Attenuated virus
  • 1996 DNA vaccines
  • The third vaccine revolution

53
DNA vaccines
  • DNA vaccines are at present experimental , but
    hold promise for future therapy since they evoke
    both humoral and cell-mediated immunity, without
    the dangers associated with live virus vaccines

54
DNA Vaccines
Gene for antigen
Muscle cell
plasmid
Muscle cell expresses protein - antibody made CTL
response
55
DNA Vaccines
  • Plasmids are easily manufactured in large
    amounts
  • DNA is very stable
  • DNA resists temperature extremes so storage and
    transport are straight forward
  • DNA sequence can be changed easily in the
    laboratory. This means that we can respond to
    changes in the infectious agent
  • By using the plasmid in the vaccinee to code for
    antigen synthesis, the antigenic protein(s) that
    are produced are processed (post-translationally
    modified) in the same way as the proteins of the
    virus against which protection is to be produced.
    This makes a far better antigen than purifying
    that protein and using it as an immunogen.

56
DNA Vaccines
  • Mixtures of plasmids could be used that encode
    many protein fragments from a virus/viruses so
    that a broad spectrum vaccine could be produced
  • The plasmid does not replicate and encodes only
    the proteins of interest
  • No protein component so there will be no immune
    response against the vector itself
  • Because of the way the antigen is presented,
    there is a CTL response that may be directed
    against any antigen in the pathogen. A CTL
    response also offers protection against diseases
    caused by certain obligate intracellular
    pathogens (e.g. Mycobacterium tuberculosis)

57
DNA Vaccines
  • Possible Problems
  • Potential integration of plasmid into host
    genome leading to insertional mutagenesis
  • Induction of autoimmune responses (e.g.
    pathogenic anti-DNA antibodies)
  • Induction of immunologic tolerance (e.g. where
    the expression of the antigen in the host may
    lead to specific non-responsiveness to that
    antigen)

58
DNA Vaccines
  • DNA vaccines produce a situation that reproduces
    a virally-infected cell
  • Gives
  • Broad based immune response
  • Long lasting CTL response
  • Advantage of new DNA vaccine for flu
  • CTL response can be against internal protein
  • In mice a nucleoprotein DNA vaccine is effective
    against a range of viruses with different
    hemagglutinins

59
Adjuvants
  • Certain substances, when administered
    simultaneously with a specific antigen, will
    enhance the immune response to that antigen.

60
Adjuvants in common use
  • Aluminium salts
  • Liposomes and immunostimulating complexes
  • Complet Freunds adjuvant is an emulsion of
    mycobacteria, oil and water
  • Incomplete Freunds adjuvant
  • Muramyl di-peptide
  • Cytokines

61
Possible action modes of adjuvant
  • By trapping antigen in the tissues, thus allowing
    maximal exposure to dendritic cells and specific
    T and B lymphocytes
  • By activating antigen-presenting cells to
    secrete cytokines that enhance the recruitment of
    antigen-specific T and B cells to the site of
    inoculation

62
Smallpox
63
Smallpox
  • Variolation
  • 1 v. 25 mortality
  • Life-long immunity
  • No drift or shift

64
Smallpox
65
Smallpox
66
polio
  • Killed virus vaccine(Salk, 1954)
  • Live attenuated oral polio vaccine( Sabin, 1957)
  • The inactivated Salk vaccines is recommended for
    children who are immunosuppressed.

67
Polio Vaccine
68
100
Inactivated (Salk) vaccine
Cases per 100,000 population United States
10
Oral vaccine
1
Reported cases per 100000 population
0.1
0.01
0.001
1950
1960
1990
1970
1980
69
Total casesSweden and Finland
10000
Killed (Salk) vaccine
1000
Reported cases
100
10
1
0
1950
1955
1960
1965
1970
1975
70
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71
Sabin Polio Vaccine
  • Attenuation by passage in foreign host
  • More suited to foreign environment and less
    suited to original host
  • Grows less well in original host
  • Polio
  • Monkey kidney cells
  • Grows in epithelial cells
  • Does not grow in nerves
  • No paralysis
  • Local gut immunity (IgA)
  • Pasteur rabies vaccine also attenuated

72
Salk Polio Vaccine
  • Formaldehyde-fixed
  • No reversion

73
Polio Vaccine
  • Why use the Sabin vaccine?
  • Local immunity Vaccine virus just like natural
    infection
  • Stopping replication in G.I. Tract stops viral
    replication TOTALLY
  • Dead Salk vaccine virus has no effect on gut
    replication
  • No problem with selective inactivation
  • Greater cross reaction as vaccine virus also has
    antigenic drift
  • Life-long immunity

74
Measles
  • Live attenuated virus grown in chick embryo
    fibroblasts, first introduced in the 1960s.
  • Etiology Measles virus
  • Incubation 8 to 12 days
  • Clinical Manifestations cough, coryza,
    conjunctivitis , erythematous maculopapular rash
  • fever ,Koplik Spots ,complictions include
    Encephalitis, Pneumonia, and SSPE
  • Treatment Supportive

75
Mumps
  • Live attenuated virus developed in the 1960s
  • MMR vaccine
  • Etiology Mumps Virus
  • Incubation 16 to 18 days
  • Clinical Manifestations
  • swelling of the salivary glands
  • complications include Meningitis, Orchitis,
    Encephalitis, and Deafness

76
rubella
  • Live attenuated virus
  • Etiology Rubella Virus
  • Incubation 14 to 21 days
  • Clinical Manifestations Congenital , cataracts
  • patent ductus arteriosus , deafness mental
    retardation , Postnatal mild disease ,
    erythematous maculopapular rash , postauricular
    lymphadenopathy transient polyarthralgias

77
Hepatitis B
  • Two vaccines are in current use
  • A serum derived vaccine
  • A recombinant vaccine
  • Etiology Hepatitis B Virus
  • Incubation 120 days (average)
  • Clinical Manifestations jaundice anorexia
  • nausea and vomiting malaise
  • complications include the development of a
    chronic carrier state with a high risk for
    Hepatocellular Carcinoma (liver cancer)

78
Hepatitis A
  • Formalin-inactivated , cell cultured-derived
    virus,

79
Yellow fever
  • The 17D strain is a live attenuated vaccine
    developed in 1937.
  • It is a highly effective vaccine which is
    administered to residents in the tropics and
    travellers to endemic areas.

80
Rabies
  • No safe attenuated strain of rabies virus has yet
    been developed for human. Vaccines in current use
    include a The neurotissue vaccine
  • b human diploid cell
    culture-derived vaccine, which is much safer.
  • There are two situation where vaccine is given
    a Post-exposure prophylaxis, followinf the bite
    of a rabid animal, Hyperimmune rabies globulin
    may also administered .
  • b Pro-exposure prophylaxis is used for
    protection of those occupation puts them at risk
    of infection with rabies.

81
Influenza
  • New vaccines are produced every year

82
Varicella-Zoster virus
  • Not licensed vaccines

83
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84
Passive Immunisation
85
Modes of immunization
  • Passive immunization - administration of
    antibody-containing serum to provide immediate,
    but temporary protection. Doesn't activate a
    lasting specific immune response.

86
Natural
  • Provides immunity for diphtheria, tetanus,
    streptococcus, rubeola (red measles), rubella
    (German measles), mumps, polio, and others.

87
Artificial
  • Often used as antitoxins for things such as black
    widow spider and snake bites, botulism, and
    tetanus. Important for some infectious diseases
    such as rabies, since it provides immediate
    protection rather than waiting the 7-10 days for
    a protective response to develop from active
    immunization.

88
Immunoglobulin
  • NormalImmune globulin
  • Hyper-immune globulin

89
NormalImmune globulin
  • Low titres of antibody to a wide range of human
    viruses
  • Hepatitis A virus infection
  • Parvovirus infection
  • Enterovirus infections (in neonates)
  • HIV-infected babies

90
Hyper-immune globulin--- high titres of antibody
to particular viruses
  • Zoster immune globulin prevention of varicella
    in immunocompromised children and neonates
  • Human rabies immunoglobulin post-exposure
    prophylaxis in an individual who has been bitten
    by a rabid animal
  • Hepatitis B immune globulinnon-immune individal
    who has been exposed to HBV
  • RSV immune globulin treatment of respiratory
    syncitial virus infections in the very young

91
Antiviral Therapy
92
Antiviral Therapy
  • Antiviral chemotherapy
  • Interferon
  • Gene therapy
  • Chinese Herbs

93
Antiviral chemotherapeutic Agents
  • Antiviral drugs are available to treat only a few
    viral diseases.
  • The reason for this is the fact that viral
    replication is so intimately associated with the
    host cell that any drug that interferes
    significantly with viral replication, is likely
    to be toxic to the host

94
Targets for chemotherapeutic agents
  • Attachment to host cell
  • Uncoating (amantadine)
  • Synthesis of viral mRNA-(interferon)
  • Translation of mRNA-(interferon)
  • Replication of viral RNA or DNA- (nucleoside
    anologues)
  • Maturation of new virus proteins-(protease
    inhibitors)
  • Budding , release

95
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96
Diseases for which effective therapy is available
  • AIDS
  • Zidovudine???? Lamivudine???? protease
    inhibitors
  • Influenza Amantadine
  • Herpes simplex virus Acyclovir
  • Varicella-Zoster virus Acyclovir
  • Cytomegalovirus Gancyclovir????, Foscarnet???
  • Respiratory syncitial virus Ribavirin????

97
Nucleotide analogues
  • Nucleotide analogues competes with normal
    nucleotide for incorporation into viral DNA or
    RNA.

98
Interferon
  • Direct antiviral effect ( prevents the infection
    of new cells) by a) degradation of viral mRNA,
    and b) inhibition of protein synthesis
  • Enhancement of the specofic immuneresponse by
    increasing the expression of MHC class I
    molecules on the surface of infected cells, the
    interferons increase the opportunity for specifif
    cytotoxic T cells to recognise and kill infected
    cells
  • Chronic hepatitis B and C virus

99
Chinese Herbs
  • ??????????????
  • ???
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