Download Chapter 17 – Procedures for Identifying Pathogens and Diagnosing

BIOL 2320
HCC-Stafford Campus
J.L. Marshall, Ph.D.
Chapter 17 – Procedures for Identifying Pathogens and Diagnosing Infections*
*Lecture notes are to be used as a study guide only and do not represent the comprehensive information you will need to know for
the exams.
17.1 An Overview of Clinical Microbiology
Basic background information on identification and diagnostic testing of how to identify unknown microbes.
The techniques used fall into three (3) categories: 1) phenotypic – lab results you can see; 2) immunologic –
lab results that use antibodies; and 3) genotypic – genetic techniques, use DNA and/or RNA. Use data from all
three to help specifically identify microbes.
Phenotypic Methods
Microscopic Morphology – use staining methods (Gram stain, spore stain, acid-fast stain) to identify bacteria
based on their reaction to the stain, along with their morphology and arrangement. This can be done before
culturing to narrow down the most likely cause of the infection.
Macroscopic Morphology – traits that you can see with your eyes, such as culture characteristics on agar
plates.
Physiological/Biochemical Characteristics – test for the presence of enzymes, use to assess metabolic and
biochemical properties of the bacterium.
Genotypic Methods
Genotypic methods use genetic material, DNA and/or RNA, to identify the bacterium. It is not necessary to
culture the bacterium, and these methods are automated.
Immunologic Methods
Microbes have antigens that stimulate the immune response by producing antibodies. Antibodies can be
tested using a blood sample. Since antibodies are specific to their antigens, this can be very determinative to
identifying the infectious agent.
On the Track of the Infectious Agent: Specimen Collection
It is important to use aseptic technique in specimen collection (figure 17.1a). Very careful collection
techniques should be used when collecting samples from areas that already have resident microbes (figure
17.1b). When collecting from areas that are normally sterile, then use of antisepsis on the skin and sterile
needle should be used. Table 17.1 summarizes common collection procedures based on the site of collection.
Special Considerations in Specimen Analysis
The results of the lab tests can be divided into two (2) categories: 1) presumptive data and 2) confirmatory
data (figure 17.2).
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BIOL 2320
HCC-Stafford Campus
J.L. Marshall, Ph.D.
17.2 Phenotypic Methods
Immediate Direct Examination of Specimen
Direct examination of a stained specimen can be rapid, and presumptive and confirmatory. Not all
“traditional” stains, like the Gram stain, work with all microbes. Direct fluorescent antibody tests are often
used with difficult to culture bacteria, like syphilis (figure 17.3).
An alternative is to use antigens that are part of the bacteria cell. Antibodies that are known to bind to these
bacterial antigens are used to test for the presence of the bacterium.
Cultivation of Specimen
Isolation Media
Based on the quantity of the bacterium present and its specific growth conditions, it may be necessary to use
selective and enrichment media to culture enough bacteria to test and make a diagnosis. Differential media
can also be used to test for fermentation patterns.
Biochemical Testing
These tests help to identify the enzymes present. The microbes are cultured in a medium that contains the
substrates. If the enzyme is present, usually a change in the color of the test medium will occur (figure 17.4).
Characterizing the growth patterns based on the biochemical tests used can help identify the causative agent
(figure 17.5).
Determining Clinical Significance of Cultures
Depending on the patient sample, such as urine, CSF or blood, the number and type of microbes present can
help determine the clinical significance.
17.3 Genotypic Methods
DNA Analysis Using Genetic Probes
Hybridization takes advantage of the unique arrangement and types of genes that are unique to each genus
and species. Hybridization of DNA and RNA uses probes to detect the presence of certain genes. Hybridization
techniques such as FISH and PFGE can be used to identify the genetic composition of microbes.
Roles of the Polymerase Chain Reaction and Ribosomal RNA in Identification
PCR can be used for DNA and RNA analysis. PCR based techniques can be used for detecting HIV, HPV, TB, and
various bacterial and viral infections. The use of rRNA to detect species for identification and/ or evolution can
be done using PCR (figure 17.7).
17.4 Immunologic Methods
The presence of antibodies in blood can be very useful in identifying microbes that cause infection. Serology is
in vitro diagnostic testing of serum. The power of serology testing is the specificity of antibodies to their
antigens. One can use patient serum (antibodies) to test against known antigens, and vice versa (figure 17.8).
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BIOL 2320
HCC-Stafford Campus
J.L. Marshall, Ph.D.
General Features of Immune Testing
Again, these tests take advantage the specificity between antibodies and antigens (figure 17.9). Specificity
tests for a particular antibody and/or antigen. Sensitivity tests the level that an antibody / antigen can be
detected.
Visualizing Antigen-Antibody Interactions
Some Ab-Ag interactions are large enough to see macroscopically and/or microscopically (figure 17.10a). For
smaller Ab-Ag interactions, the use of fluorescent dyes and radioactive tracers can be used. Ab can be used in
a titer to determine the concentration of Abs in a patient sample (figure 17.10b).
Agglutination and Precipitation Reactions
Agglutination- antigens are whole cells, like RBCs or bacteria.
Precipitation- antigens are soluble molecules.
In both cases when the concentration of Ab and Ag are optimal, an insoluble aggregate is formed that settles
out of solution.
Agglutination Tests
The Ab crosslinks the Ag to form visible clumps. This test is done on the ABO/Rh blood groups. Other tests that
use agglutination are: Widal test for salmonellosis, RPR for syphilis, and latex agglutination, which is used for
pregnancy, Candida, and staphylococci.
Precipitation Tests
The soluble Ag is precipitated by the Ab. This is usually done in a test tube. An example is the VDRL test, which
is used to detect antibodies to syphilis. Another method id the double diffusion test. Bands in a soft agar gel
indicate the Ab-Ag interaction (figure 17.11).
The Western Blot for Detecting Proteins
The Western blot test involves proteins that are separated by electrophoresis, then detected with Ab (figure
17.12). This test is used to confirm HIV status.
Complement Fixation
Complement fixation test is conducted in two (2) stages (figure 17.13):
stage 1 – the patient’s antibodies bind complement and the test antigen;
stage 2 - the complement is not available to bind the test rbcs, no lysis of rbc = Ab present
-> this is a positive reaction.
stage 1 – the patient does not have antibodies for the infectious agent, therefore complement is free in
solution
stage 2- the complement can bind to the rbcs and cause lysis, lysis of rbcs = no Ab present
-> this is a negative reaction.
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BIOL 2320
HCC-Stafford Campus
J.L. Marshall, Ph.D.
Fluorescent Antibody and Immunofluorescent Testing
The key component to this testing is a fluorescent antibody (FAB), a monoclonal antibody that has a
fluorescent dye attached. There are two (2) ways to use FABs: 1) direct testing: unknown Ag + FAB = visible
fluorescence (figure 17.14a); 2) indirect testing: i) known Ag + Ab in serum + FAB = visible fluorescence, or ii)
known Ag + no Ab in serum + FAB = no visible fluorescence (figure 17.14b).
17.5 Immunoassays: Tests of Great Sensitivity
Radioimmunoassay (RIA)
Label Ab or Ag with radioactive isotope, can find minute amounts in the sample. The more radioactivity after
exposure to the sample, the less likely the Ab or Ag is present. The less radioactivity after exposure to the
sample, the more likely the Ab or Ag is present.
Enzyme-Linked Immunosorbent Assay (ELISA)
The ELISA this test uses an enzyme-antibody complex with a color tracer for Ab-Ag interactions (figure 17.15).
The indirect ELISA the test serum that contains the Ab attaches to a known Ag. A second Ab with an enzyme
label attaches to the first serum Ab, and a color reaction is detected (figure 17.15a). The capture ELISA a
known Ab is absorbed to the well, a test Ag is added, then the second enzyme-Ab complex is added. A color
reaction indicates a positive result (figure 17.15c).
In Vivo Testing
Use the body itself to test for the presence of Ab. One such test is the tuberculin test for TB infections. A
positive reaction is a raised and swollen red bump after 72 hours.
17.6 Viruses as a Special Diagnostic Case
Viruses are not cells, therefore, some of the techniques previously mentioned will not always work on them.
Other methods are used to test for the presence of viruses. See figure 17.16.
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