Chapter 17
Diagnosing Infections
How to Identify the Disease
Direct testing (microscopic, immunological, genetic methods)
Time required: from few minutes
Cultivation of microorganisms
Time required: few days to few weeks (Mycobacterium tuberculosis)
Based on symptoms (athletes foot)
How to Identify Unknown Bacteria
Phenotypic methods
Macroscopic morphology
Microscopic morphology
Physiological/Biochemical Characteristics
Genotypic methods
Culturing not necessary
Results obtained quickly
Immunological methods
Looking for the specific antibody in patients blood
Specimen Collection
Can be collected by a member of a clinical team or by a patient
Aseptic procedures should be followed
Avoid taking samples of normal biota (e.g. saliva when taking throat swab)
Samples should be promptly transported to the lab
Immediate Direct Examination of Specimen
Direct microscopic observation
Fresh
Stained (Gram staining, acid fast staining)
Direct fluorescence antibody test
Antibody labeled with a fluorescent dye
Cultivation of Specimen
A dominant organism has to be isolated into a pure culture
The isolated microorganism is characterized by
Microscopic morphology
Staining reaction (Gr+ or Gr-)
Cultural appearance
Motility
Oxygen requirements
Biochemical characteristics
Biochemical tests
Differentiation among the bacterial species - Enzymatic activity (fermentation of glucose, lactose , production H2S, presence of catalase, use of citric acid as sole carbon source .)
Rapid biochemical systems for identification of medically important bacteria have been developed
Disadvantage of the method: mutation can result in strains with different characteristics
Phage typing
Identifies which phage affects specific bacterial strain
Can distinguish strains within one species
Enables tracking the source of the outbreak
Hybridization with a probe
The method used to detect specific nucleotide sequence in an unknown sample by using a gene probe
Gene probe is a short segments of DNA of a known sequence
A probe carries a radioactive label
DNA typing of restriction fragment length polymorphism (RFLP)
DNA from M. tuberculosis from 17 patients is cut with a restriction enzyme
By comparing the pattern of DNA fragments it is possible to determine which patients were infected with the same bacterial strain
Diagnostic Immunology
Use of immunological methods to identify pathogenic microbes
Main characteristic of this method Specificity- specific antibody will react only with a specific antigen
Either antigen or antibody can be used
Reactions are visible with a naked eye
Types of Immunological Tests
Precipitation tests
Agglutination tests
Immunodiffusion
Complement fixation
Fluorescence-antibody technique
Immune assay
Agglutination reactions
Particulate antigens (microbial cells) react with antibodies forming visible aggregates
Precipitation reaction
Soluble antigens react with antibodies forming large molecular aggregates visible precipitate
There are two techniques:
Precipitation in liquid
Precipitation in agar
Precipitation in agar double diffusion test
The wells in the agar are filled with test antigens and various antibodies
They diffuse in agar
The positive reaction is the line precipitate between two wells
Used for the detection of antibodies to syphilis
Quantification of antibodies in the serum
Determining the titer
Patients serum is serially diluted
Mixed with antigen
Incubate and centrifuge
Tubes inspected for clumps
Titer is the dilution of the last tube that shows agglutination
A change of the titer over time indicates whether the disease is subsiding or advancing
Complement fixation test
Components needed for this test
Antibody
Antigen
Complement
Sensitized sheep red blood cells
Conducted in two stages
1st stage
test antigen allowed to react with the known antibody in the absence of complement
Complement added
If antibody and antigen made a complex, then they will fix the complement making it unavailable
2nd stage of testing
Sheep RBC containing cytolysins are mixed with the content of the stage 1 tube
Hemolysis (clearing of the solution) is observed
If the complement is fixed to antigen-antibody complex no hemolysis will occur the reaction is positive
Neutralization reaction
Clumping of red blood cells
Some viruses (measles and influenza) can clump the red blood cells
Not an antigen-antibody reaction
Antibodies can block exotoxin or a virus.
This is used for diagnosis of influenza, measles, mumps, etc. by viral hemagglutination inhibition test.
Procedure:
Patients serum + erythrocytes + a virus suspension
If the serum contains the antibodies against a specific virus hemagglutination will not occur.
Immunofluorescence testing
Direct Fluorescent-antibody test
Antibodies are tagged with the fluorescent antibody
Sample is flooded with fluorescently labeled antibody
If the tested sample has receptors for the particular antibody they will conjugate
Those cells will fluoresce when illuminated with UV light. (Fluorescent microscopy).
Enzyme-linked immunosorbent assay (ELISA)
A known antigen is absorbed onto a surface of the well
An unknown antibody (serum) is added
Well is rinsed
An antibody with an attached enzyme that can react with the unknown test antibody is added
Well is rinsed
The substrate for the enzyme is placed in the well
If the enzyme-linked antibody recognizes the unknown antibody the color will develop
The intensity of the color is proportional to the amount of the unknown antibody
Use of ELISA
Indirect ELISA is used for screening for the antibodies to
HIV
Hepatitis A and C
Cholera
Helicobacter (gastric ulcers)
Sandwich ELISA is used for detection of
Hantavirus
Rubella virus
Toxoplasma (protozoan)