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Clinical Microbiology and Immunology

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Title: Clinical Microbiology and Immunology


1
Chapter 32
  • Clinical Microbiology and Immunology

2
Specimens
  • Clinical microbiologist
  • major function is to isolate and identify
    microbes from clinical specimens rapidly
  • Clinical specimen
  • portion or quantity of human material that is
    tested, examined, or studied to determine the
    presence or absence of specific microbes

3
Working with Specimens
  • Safety concerns
  • Standard Microbiological Practices have been
    established by the Centers for Disease Control
    and Prevention (CDC)
  • Specimen should
  • represent diseased area and other appropriate
    sites
  • be large enough for carrying out a variety of
    diagnostic tests
  • be collected in a manner that avoids
    contamination
  • be forwarded promptly to clinical lab
  • be obtained prior to administration of
    antimicrobial agents, if possible

4
Identification of Microorganisms from Specimens
  • Preliminary or definitive identification of
    microbe based on numerous types of diagnostic
    procedures
  • microscopy
  • growth and biochemical characteristics
  • immunologic tests
  • bacteriophage typing
  • molecular methods

5
Collection
  • numerous methods used
  • choice of method depends on specimen

6
Immunofluorescence
  • process in which fluorescent dyes are exposed to
    UV, violet, or blue light to make them fluoresce
  • dyes can be coupled to antibody molecules with
    changing antibodys ability to bind a specific
    antigen
  • can be used as direct fluorescent-antibody (FA)
    technique or indirect fluorescent-antibody (IFA)
    technique assay

7
FA technique
Figure 32.2a
8
IFA technique
Figure 32.2b
9
Growth and Biochemical Characteristics
  • techniques used depend on nature of pathogen
  • for some pathogens, culture-based techniques have
    limited use

10
Viruses
  • Identified by
  • isolation in living cells
  • immunodiagnostic tests
  • molecular methods
  • replication in culture detected by
  • cytopathic effects
  • morphological changes in host cells
  • hemadsorption
  • binding of red blood cells to surface of infected
    cells

11
Fungi
  • Cultures used to recover fungus from patient
    specimens
  • growth medium depends on type(s) of fungus being
    isolated
  • Identification
  • direct microscopic (fluorescence) examination
  • immunofluorescence
  • serological tests (for some)
  • rapid identification methods (most yeasts)

12
Bacteria
  • Most bacteria
  • culturing involves use of numerous kinds of
    growth media
  • can provide preliminary information about
    biochemical nature of bacterium
  • additional biochemical tests and staining used
    following isolation
  • some bacteria are not routinely cultured
  • rickettsias, chlamydiae, and mycoplasmas
  • identified with special stains, immunologic
    tests, or molecular methods such as PCR

13
Rapid Methods of Identification
  • manual biochemical systems
  • mechanized/automated systems
  • immunologic systems

14
Biosensors
  • based on the linkage of traditional
    antibody-based detection systems to sophisticated
    reporting systems
  • can be based on
  • microfluidic antigen sensors
  • real time PCR
  • highly sensitive spectroscopy systems
  • liquid crystal amplification of microbial immune
    complexes

15
Molecular Methods and Analysis of Metabolic
Products
  • several methods widely used
  • examples include
  • nucleic acid probes
  • ribotyping
  • genomic fingerprinting

16
Genomic Fingerprinting
  • characterizes bacteria based on restriction
    endonuclease digestion of DNA
  • plasmid fingerprinting uses number of plasmids,
    their molecular weight, and restriction digestion
    pattern

Figure 32.5
17
Immunological Techniques
  • Detection of antigens or antibodies in specimens
  • especially useful when cultural methods are
    unavailable or impractical or antimicrobial
    therapy has been started

18
Clinical Immunology Serotyping
  • Clinical Immunology
  • many antibody-antigen interactions that occur in
    vivo can also be used under controlled laboratory
    conditions for (in vitro) diagnostic testing
  • Serotyping
  • use of serum antibodies to detect and identify
    other molecules
  • can be used to differentiate serovars or
    serotypes of microbes that differ in antigenic
    composition of a structure or product

19
Agglutination
  • Agglutinates
  • visible clumps or aggregates of cells or
    particles
  • e.g., Widal test
  • diagnostic for typhoid fever
  • e.g., latex agglutination tests
  • pregnancy test
  • e.g., viral hemagglutination
  • can be used to indicate the presence of
    virus-specific antibodies

20
Agglutination Tests
titer reciprocal of highest dilution positive
for agglutination
Figure 32.8
21
Enzyme-Linked Immunosorbent Assay (ELISA)
  • can be used to detect antigens or antibodies in a
    sample
  • test involves the linking of various label
    enzymes to either antigens or antibodies
  • two basic methods used
  • direct immunoabsorbant assay
  • indirect immunoabsorbant assay

22
Immunoblotting (Western Blot)
  • procedure
  • proteins separated by electrophoresis
  • proteins transferred to nitrocellulose sheets
  • protein bands visualized with enzyme-tagged
    antibodies
  • sample uses
  • distinguish microbes
  • diagnostic tests
  • determine prognosis for infectious disease

23
Radioimmunoassay (RIA)
  • purified antigen labeled with radioisotope
    competes with unlabeled standard for antibody
    binding
  • amount of radioactivity associated with antibody
    is measured

24
Bibliography
  • Lecture PowerPoints Prescotts Principles of
    Microbiology-Mc Graw Hill Co.
  • http//en.wikipedia.org/wiki/Scientific_method
  • https//files.kennesaw.edu/faculty/jhendrix/bio334
    0/home.html
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