Microbiology: A Systems Approach, 2nd ed.

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Microbiology A Systems Approach, 2nd ed.

• Chapter 17 Diagnosing Infections

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17.1 Preparation for the Survey of Microbial
Diseases

• Methods used to identify bacteria to the level of
  genus and species
• Phenotypic methods
• Morphology
• Physiology or biochemistry
• Immunologic method
• Serological analysis
• Genotypic techniques
• More and more often used as a sole resource for
  identifying bacteria

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Phenotypic Methods

• Microscopic morphology
• Macroscopic morphology
• Physiological/Biochemical characteristics
• Chemical analysis

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Microscopic Morphology

• Cell shape and size
• Gram stain reaction
• Acid fast reaction
• Special structures

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Macroscopic Morphology

• Colony appearance
• Speed of growth
• Patterns of growth

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Physiological/Biochemical Characteristics

• Traditional mainstay of bacterial identification
• Diagnostic tests for determining the presence of
  specific enzymes and assessing nutritional and
  metabolic activities
• Examples
• Fermentation of sugars
• Capacity to metabolize complex polymers
• Production of gas
• Presence of enzymes
• Sensitivity to antimicrobic drugs

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Chemical Analysis

• Analyzing the types of specific structural
  substances that the microorganism contains
• Examples
• Chemical composition of peptides in the cell wall
• Lipids in membranes

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Genotypic Methods

• Primary advantage over phenotypic methods
  actually culturing the microorganisms is not
  always necessary
• Also are increasingly automated with results
  obtained very quickly

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Immunologic Methods

• Antibody response to antigens
• Blood testing- often easier than testing for the
  microbe itself
• Laboratory kits available for immediate
  identification of a number of pathogens

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17.2 On the Track of the Infectious Agent
Specimen Collection

• The success of identification and treatment
  depends on how specimens are collected, handled,
  and stored
• General aseptic procedures must be used

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Figure 17.1
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Overview of Laboratory Techniques

• Direct tests using microscopic, immunologic, or
  genetic methods
• Cultivation, isolation, and identification of
  pathogens using a wide variety of general and
  specific tests
• Results of specimen analysis entered in a summary
  patient chart

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Figure 17.2
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Figure 17.3
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17.3 Phenotypic Methods

• Immediate Direct Examination of Specimen
• Gram stain
• Acid-fast stain
• Direct fluorescence antibody (DFA) tests
• Direct antigen testing

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Figure 17.4
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Cultivation of Specimen

• Isolation media
• Biochemical testing
• Carbohydrate fermentation (acid and/or gas)
• Hydrolysis of gelatin, startch, and other
  polymers
• Enzyme actions such as catalase, oxidase, and
  coagulase
• By-products of metabolism

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Figure 5
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Figure 17.6
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Miscellaneous Tests

• Phage typing
• Animal inoculation
• Antimicrobial sensitivity

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Determining Clinical Significance of Cultures

• Is an isolate clinically important?
• How do you decide whether it is a contaminant or
  part of the normal biota?
• Possible criteria
• Number
• Repeated isolation of a relatively pure culture
  of any microorganism

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17.4 Genotypic Methods

• DNA Analysis Using Genetic Probes
• Hybridization- can identify a bacterial species
  by analyzing segments of its DNA
• Small fragments of single-stranded DNA or RNA
  called probes
• Known to be complementary to the specific
  sequences of DNA from a particular microbe
• Unknown test DNA from cells is bound to blotter
  paper
• Add probes to blotter
• Observe for signs that the probes have become
  fixed to the test DNA

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Figure 17.7
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Nucleic Acid Sequencing and rRNA Analysis

• Comparison of the sequence of nitrogen bases in
  rRNA
• Effective for differentiating general group
  differences
• Can be fine-tuned to identify at the species
  level

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Polymerase Chain Reaction

• Rapid identification of pathogens
• Developed for a wide variety of bacteria,
  viruses, protozoa, and fungi
• Biosensor

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17.5 Immunologic Methods

• Characteristics of antibodies can reveal the
  history of a patients contact with
  microorganisms or other antigens
• Serological testing
• Serology the branch of immunology that
  traditionally deals with in vitro diagnostic
  testing of the serum

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Figure 17.8
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General Features of Immune Testing

• Strategies
• Agglutination
• Precipitation
• Immunodiffusion
• Complement fixation
• Fluorescent antibody tests
• Immunoassay tests
• Specificity and sensitivity

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Figure 17.9
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Visualizing Antigen-Antibody Interactions
Figure 17.10
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Agglutination and Precipitation Reactions

• Agglutination antigens are whole cells such as
  red blood cells or bacteria with determinant
  groups on the surface
• Precipitation the antigen is a soluble molecule

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Agglutination Testing

• Antibodies cross-link the antigens to form
  visible clumps
• Performed routinely to determine ABO and Rh blood
  types
• Widal test tube agglutination test for
  diagnosing salmonelloses and undulant fever
• Rapid plasma regain (RPR) test tests for
  antibodies to syph8ilis
• Weil-Felix reaction diagnoses ricketsial
  infections
• Latex agglutination tests tiny latex beads with
  antigens affixed

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Precipitation Tests

• The soluble antigen is precipitated by an
  antibody
• Reaction is observable as a cloudy or opaque zone
  at the point of contact
• VDRL (Veneral Disease Research Lab) test
• Double diffusion (Ouchterlony) method
• Immunoelectrophoresis

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Figure 17.11
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Figure 17.12
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The Western Blot for Detecting Proteins

• Involves electrophoretic separation of proteins
  followed by an immunoassay to detect those
  proteins
• Counterpart of the Southern blot test
• Test material is electrophoresed in a gel to
  separate out particular bands
• Gel transferred to a special blotter that binds
  the reactants in place
• Blot developed by incubating it with a solution
  of antigen or antibody labeled with radioactive,
  fluorescent, or luminescent labels

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Figure 17.13
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Complement Fixation

• Lysin or cytolysin an antibody that requires
  complement to complete the lysis of its antigenic
  target cell

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Figure 17.14
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Miscellaneous Serological Tests

• Treponema pallidum immobilization (TPI) test
• Toxin neutralization tests
• Serotyping
• Quellung test

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Flurorescent Antibodies and Immunofluorescence
Testing

• Direct testing an unknown test specimen or
  antigen is fixed to a slide and exposed to a
  fluorescent antibody solution of known
  composition
• Indirect testing the fluorescent antibodies are
  antibodies made to react with the Fc region of
  another antibody

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Figure 17.15
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Immunoassays

• Extremely sensitive methods that permit rapid and
  accurate measurement of trace antigen or antibody
• Radioactive isotope labels
• Enzyme labels
• Sensitive electronic sensors

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Radioimmunoassay (RIA)

• Antibodies or antigens labeled with a radioactive
  isotope used to pinpoint minute amounts of a
  corresponding antigen or antibody
• Compare the amount of radioactivity present in a
  sample before and after incubation with a known,
  labeled antigen or antibody
• Large amounts of a bound radioactive component
  indicate that the unknown test substance was not
  present
• Radioimmunosorbent test (RIST)
• Radioallergosorbent test (RAST)

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Enzyme-Linked Immunosorbent Assay (ELISA)

• Enzyme-antibody complex that can be used as a
  color tracer for antigen-antibody reactions
• Indirect
• Direct

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Figure 17.16
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Tests that differentiate T Cells and B Cells

• Mix with untreated sheep red blood cells
• T cells bind RBCs into a rosette formation

Figure 17.17
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In Vivo Testing

• Tuberculin test
• Other diagnostic skin tests

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A Viral Example
Figure 17.18