Clinical Microbiology and Immunology

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Chapter 32

• Clinical Microbiology and Immunology

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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

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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

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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

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Collection

• numerous methods used
• choice of method depends on specimen

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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

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FA technique
Figure 32.2a
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IFA technique
Figure 32.2b
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Growth and Biochemical Characteristics

• techniques used depend on nature of pathogen
• for some pathogens, culture-based techniques have
  limited use

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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

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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)

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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

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Rapid Methods of Identification

• manual biochemical systems
• mechanized/automated systems
• immunologic systems

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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

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Molecular Methods and Analysis of Metabolic
Products

• several methods widely used
• examples include
• nucleic acid probes
• ribotyping
• genomic fingerprinting

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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
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Immunological Techniques

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

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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

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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

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Agglutination Tests
titer reciprocal of highest dilution positive
for agglutination
Figure 32.8
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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

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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

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Radioimmunoassay (RIA)

• purified antigen labeled with radioisotope
  competes with unlabeled standard for antibody
  binding
• amount of radioactivity associated with antibody
  is measured

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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