Laboratory diagnosis of infectious diseases Flashcards
Detection of pathogenic agents:
- Microscopic visualisation of pathogens in clinical material
- Detection of the growth of microorganisms in the laboratory
Identification is based on phenotypic characteristics such as:
- fermentation profiles of bacteria
- cytopathic effects in tissue culture for viral agents
- microscopic morphology for fungi or parasites
What is the first tool in diagnosing microbial disease?
Microbial cultures
What is the sample that is obtained from the patient tested for?
The sample is obtained from the infected individual and tested for the presence of INFECTIOUS AGENT OR MICROBE that is capable of growing in specific media.
- It is critical to isolate the infectious agent in a pure culture containing only the infectious bacteria
What is the most common method to isolate individual cells and produce a pure culture?
The most common method to isolate individual cells and produce a pure culture IS TO PREPARE A STREAK PLATE
How is the streak plate method done?
- The streak plate method is a way to physically separate the microbial population, and it is done by spreading the inoculate back and forth with an inoculating loop over the solid agar plate.
- Upon incubation, colonies will arise and single cells will have been isolated from the biomass
Limitation of the conventional methods:
- lenghty, time consuming
- associated with risk
- culturing of certain organisms like viruses, fungi, or parasites may not be possible
- culture may be negative due to prior antimicrobial therapy
- requires sophisticated labs (impossible in all laboratories e.g. Mycobacteria)
The laboratory diagnosis of infection - others:
- Serological identification/immunological identification of Ags or Abs
- Nucleic acid based/Molecular biology techniques
The laboratory diagnosis of infection - others - ADVANTAGES:
(- Serological identification/immunological identification of Ags or Abs
- Nucleic acid based/Molecular biology techniques)
- provide early diagnosis
- important for uncultivable organisms in the laboratory
- useful in differential diagnosis of certain disease
- useful to measure the antibody level
Biological signal:
signal generated by detection of a material that can be reproducibly differentiated from substances present in the sample
Key issue:
are distinguishing it from background noise and translating it into meaningful information
Useful materials for detection of biologic signals applicable to clinical microbiology include:
- structural components of bacteria, fungi, and viruses
- specific antigen
- metabolic end products
- unique DNA or RNA base sequences
- enzymes
- toxins or other proteins
- surface polysaccharides
What is a detector?
A detector is used to sense a signal and discriminate between that signal and background noise
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Direct detection:
Direct detection refers to detection of pathogens without the use of culture
Direct detection examples:
- microscopy and staining
- macroscopy antigen detection
- detection of pathogenic agents by serologic methods
Wet mount:
the simplest method of microscopic evaluation
What is the simplest method of microscopic evaluation?
wet mount
Example 1 of Wet Mount:
CSF examination for the presence of Cryptocosccus neoformans- with India ink
Example 2 of Wet Mount:
Wet Mount with dark-field illumination - used to detect spirochetes in genital lesions or Borrelia and Leptospira in blood
Stains that are used to see bacteria:
- gram´s stain
- acid-fast stain
- fluorochrome stain
- immunofluorescent stain
Gram´s stain:
differentiates between organisms with thick peptidoglycan cell walls (gram-positive), and outer membranes that can be dissolved with alcohol or acetone (gram-negative)
Morphology and Gram´s stain characteristics often can be used to categorise stained organisms into groups such as…….
- streptococci
- staphylococci
- clostridia
Acid-fast stain:
The acid-fast stain identifies acid-fast bacteria (eg. Mycobacteria) by their retention of carbol fuschin dye after acid/organic solvent disruption
Where can we apply acid-fast stain?
The acid-fast stain is applied to sputum, other fluids, and tissue samples when Mycobacterium species are suspected.
What is the alternative method for detection of acid-fast bacteria?
auramine-rhodamine fluorescent dye stain
Fluorochrome stains example:
Acridine orange
Fluorochrome stains:
Fluorochrome stains are used to identify white blood cells, yeast, and bacteria in body fluids.
- Capsular, flagellar or spore stains are used for identification or demonstration of characteristic structures
Immunofluorescent stains division:
- direct
- indirect
Direct Immunofluorescent stain:
The direct immunofluorescent antibody technique uses antibody coupled to fluorescent compound (eg. Fluorescein) and directed at a specific antigen target to visualise organisms
Indirect Immunofluorescent stain:
- The indirect immunofluorescent antibody technique, an unlabeled (target) antibody binds a specific antigen.
- The specimen is then stained with a fluorochrome labeled antibody directed at the target antibody.
- Because each unlabeled target antibody attached to the appropriate antigen has multiple sites for attachment of the second antibody, the visual signal is amplified.
Macroscopic antigen detection examples:
- Latex agglutination assays
- EIAs
Latex agglutination assays and EIAs:
- Latex agglutination assays and EIAs are rapid and inexpensive methods for identifying organisms, extra cellular toxins, and viral agents by means of protein and polysaccharide antigens.
- Antibodies coupled to a reporter (such as latex particles or an enzyme) are used for detection of antibody-antigen binding reactions.
Microscopy and staining examples:
- Fluorochrome stains (eg. Acridine orange)
- Immunofluorescent stains
Give example of fluorescent compound:
- Fluorescein
What organisms have an outer membranes that can be dissolved with alcohol or acetone?
gram-negative
What organisms have thick peptidoglycan cell walls ?
gram-positive
Gove example of acid-fast bacteria?
Mycobacteria
Serologic method definition:
Measurement of serum antibody provides an indirect marker for past or current infection with a specific pathogens agent.
What can we use serological methods for?
Serologic methods can be used to determine whether an individual has protective antibody levels or is infected by a specific pathogen
Conventional serological methods:
- Precipitation
- Agglutination
- Haemagglutination
- Haemagglutination inhibition
- Complement fixation test
- Fluorescent antibody test
What are Precipitation reactions?
Precipitation reactions are serological assays for the detection of immunoglobulin levels from the serum of a patient with infection.
What are Precipitation reactions are based on?
Precipitation reactions are based on the interaction of antibodies and antigens.
What are Precipitation reactions are based on - in more detail?
- They are based on two soluble reactants that come together to make one insoluble product, the precipitate.
- These reactions depend on the formation of lattices (cross-links) when antigen and antibody exist in optimal proportions.
- Excess of either component reduces lattice formation and subsequent precipitation.
Where are Precipitation assays are performed in?
Precipitation assays are performed in semi-solid media such as agar or agarose where antibodies and antigens can diffuse toward one another and form a visible line of precipitation.
There are several precipitation methods applied in the diagnostic laboratory. These include:
- single
- double
- electroimmunodiffusion.
What are the most widely used gold standard precipitation methods?
The most widely used gold standard precipitation methods are Ouchterlony test and Mancini test.
What are are Ouchterlony test and Mancini test?
The most widely used gold standard precipitation methods are Ouchterlony test and Mancini test.
(?) ………Sensitive can detect as little as 1 ug of protein
Eg. VDRL test for syphilis
What are Agglutination reactions used for?
Agglutination reactions are used to assess the presence of antibodies in a specimen by mixing it with particulate antigens.
What does the Agglutination reactions produce?
Agglutination reactions produce visible aggregates of antibody - antigen complexes when antibodies or antigens are conjugated to a carrier.
Describe the carrier used in Agglutination methods:
Carriers used in agglutination methods could be artificial (e.g., latex ) or biological (e.g., erythrocytes ).
Give example of artificial carrier used in agglutination method:
latex
Give example of biological carrier used in agglutination method:
erythrocytes
What happens ion the agglutination test?
Conjugated particles are reacted with patient serum presumably containing antibodies.
What is the endpoint of the agglutination test and what do we observe?
The endpoint of the test is the observation of clumps resulting from that antigen- antibody complex formation.
What is the quality of the result of the agglutination test determined by?
The quality of the result is determined by the time of incubation with the antibody source, amount and avidity of the antigen conjugated to the carrier, and conditions of the test environment (e.g, pH and protein concentration).
Various methods of agglutination are used in diagnostic immunology and these include:
- latex agglutination
- flocculation tests
- direct bacterial agglutination
- hemagglutination.
How is it in Latex agglutination?
- In latex agglutination, many antibody molecules are bound to latex beads (particles), which increases the number of antigen-binding sites.
- If an antigen is present in a test specimen, it will bind to the antibody and form visible, cross-linked aggregates.
- Latex agglutination can also be performed with the antigen conjugated to the beads for testing the presence of antibodies in a serum specimen.
How is it in Flocculation tests?
Flocculation tests are designed for antibody detection and are based on the interaction of soluble antigens with antibodies, producing a precipitate of fine particles that can be seen with the naked eye.
How is the Direct bacterial agglutination?
- Direct bacterial agglutination uses whole pathogens as a source of antigen.
- It measures the antibody level produced by a host infected with that pathogen.
- The binding of antibodies to surface antigens on the bacteria results in visible clumps.
How is the Hemagglutination?
Hemagglutination uses erythrocytes as the biological carriers of bacterial antigens, and purified polysaccharides or proteins for determining the presence of corresponding antibodies in a specimen.
What is Complement fixation?
Complement fixation is a method that demonstrates antibody presence in patient serum
What are the key points in complement fixation?
- Complement fixation method is more demanding than other systems used to detect antibodies and has been replaced by more sensitive techniques.
- Complement fixation requires several elements mixed together in optimum concentrations.
- The indicator system for the complement fixation assay is sheep red blood cells bound to anti-sheep immunoglobulin G.
How many complement fixation test consists of?
The complement fixation test consists of two components
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What are Fluorescent antibodies?
Fluorescent antibodies are antibodies that have been tagged with a fluorescent compound to facilitate their detection in the laboratory.
Key Points about Fluorescent antibody test:
- Fluorescent labeling of antibodies is used in place of radioisotopes and enzymes to enhance the sensitivity and specificity of immunological tests.
- Fluorescent antibodies can be used to stain proteins from patient serum or tissue sections fixed on a slide or live cells in suspension.
- Fluorescent antibodies can be detected with a fluorescent microscope or a flow cell sorter.
What is Enzyme-linked immunosorbent assay (ELISA)?
Enzyme-linked immunosorbent assay (ELISA) is a solid-phase enzyme immunoassay used to detect the presence of a substance in solution.
ELISA =
Enzyme-linked immunosorbent assay
Is ELISA a quantity or quality technique?
ELISA is a quantitative technique that measures serum concentration of antigens, antibodies, and allergens.
What does the standard ELISA test use?
Standard ELISA uses antibody-antigen-antibody trapping principle with the second antibody coupled to an enzyme.
- If the complex is formed, the enzyme converts a clear solution into a colored one that can be measured with a spectrophotometer.
What is ELISA performed in?
ELISA is performed in a muti-well microtiter plate.
- In addition to the test solution, standard solutions are added with known antigen concentration.
- These solutions will be used to infer the concentration of the antigen being tested.
What is Immunoblot?
Immunoblot is a technique for analyzing proteins via antigen-antibody specific reactions.
Give example of Immunoblot:
Western blot analysis
What happens in Immunoblot techniques?
In immunoblot techniques such as Western blot analysis, proteins are separated by electrophoresis and transferred onto nitrocellulose sheets, then are identified by their reaction with labeled antibodies.
How does the Electrophoresis work?
IMMUNOBLOT TECHNIQUES
Electrophoresis uses an electric current to separate proteins based on their size.
- Big proteins migrate slower and are represented by the highest bands on the blot, while small proteins migrate faster and are indicated by the lowest bands on the blot.
Why are Immunoblot assays performed?
Immunoblot assays are usually performed to confirm results obtained by other techniques such as ELISA.
What is the most common Identification method?
classic biochemical phenotyping
Identification methods include:
classic biochemical phenotyping (most common),
as well as more sophisticated methods such as mass spectrometry, gas chromatography,
and nucleic acid tests.
Biochemical phenotyping:
Biochemical phenotyping biochemical tests are also used to help in microbial disease diagnosis.
- They will specifically test for metabolic and enzymatic products that an infectious agent may use.
- Biochemical tests will also test for fermentation products, acids, alcohol or gases that may be products of metabolic pathways.
…..
- Many common organisms can be identified on the first day of growth.
- Other organisms particularly gam- negative bacteria, require more extensive testing, either manual or automated.
