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 (athlete’s 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 patient’s 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

•         Patient’s 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:

•         Patient’s 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)

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