Diagnosis of Infection Flashcards
What are the 2 ways that a pathogen can be identified?
- identification of the actual pathogen
- identification of the host response to the pathogen
- ex. tuberculosis can be found in the lungs, or antibodies for tuberculosis can be found in serology
Which gene can be isolated that is known to cause methicillin resistance?
MECAG gene
What two pieces of information is important when identifying a microorganism?
- presence of that microorganism
- susceptibility and the presence of a resistance gene
In what cases is it most useful to detect the specific antibodies to a pathogen rather than the actual pathogen?
- pathogens that cannot be cultivated/ takes a long time to cultivate (tuberculosis is mostly identified via presence
- high risk group pathogens
- retrospective diagnosis
How does PCR work?
you design a primer that has a specific gene within the organism- you want to design a primer that is specific to the neisseria menigitis -> needs to be specific to the certain species – need to make primer that target specific resistance genes
- if you have a primer that targets a specific gene, you can determine methacillin resistance/specific
What are the advantages of non-culture methods?
- fast
- less labor intensive
- suitable for organisms that cannot be cultured in the lab
What are some examples of non-culture methods?
- microscopy
- immunodiagnostics
- molecular diagnostics
What is the main disadvantage of using an electron microscope?
- cannot image live cells
What is resolution?
- the minimum distance that you can distinguish two objects as two objects
What are the four steps of the gram stain?
- flood with crystal violet
- flood with iodine
- flood with ethanol
- flood with safarin
What piece of information does the gram stain give you?
- tells you information about the structure of the cell wall of the pathogen
- tells you the morphology of the bacteria (any stain will do this, but only the gram stain will tell you about the cell wall)
Acid fast cells are ______
very difficult to stain - cannot wash these cells with acid after they have been stained- if you do the organism will just retain the dye
- acid fast bacteria cannot retain the gram stain - if you cannot find the microorganism after doing a test then you will most likely have an acid fast bacteria
Describe florescent staining?
- naturally florescent
- stained with florescent dyes
- many of the florescent methods empty antibodies that tag specific antigens on the cell surface- these antibodies are tagged with florescent materials
- can use this method to detect even one cell in the specimen, because the signal is that strong
Describe electron microscopy
Gives a resolution of 0.1 to 1.0 nm
- uses an electron beam instead of light
- uses magnets instead of lenses to create an image
- the specimen has to be very thin to work
- shoes the surface of the organism opposed to the inside of the specimen
- can show viruses even
- not usually used in labs
In what cases is imumunodetection used?
- used when you need a response much faster than you would get with just culturing
- specific antibody coated onto a latex bead shows visible clumping
- unusually used when bacterial meningitis is suspected - used with Strep pneumoniae, haemophilic influenzae, neisseria meningiditis in CSF
- can sometimes lead to false positives
What does ELISA allow you to do?
- allows you to quantify the pathogen, not just be able to tell if its present or not
- the secondary antibodies that are produced are labeled with an enzyme
- ALLOWS YOU TO TEST FOR ANTIBODIES FOR THE PATHOGEN
How do you quantify ELISA?
- can use spectrophotometry - more antibodies, more antibodies binding to more antigens and therefore there is more colour being produced
- we would want this assay more than the other one because it allows us to use the same secondary antibodies to detect any human antibodies
What is the process of using ELISA?
- need to add the antigen to a test of the blood
- if the antibody levels are high, the antibodies and antigen with agglutinate
- if there is an secondary antibody complex, you can put a tag on it to detect the antibodies in the blood
- tagging the secondary antibody is very critical- it will light it up
- the secondary antibody will bind to the Fc region of the antigen
What is ELISA quantified with?
can quantify this with a spectrophotometer which can quantify the intensity of the colour
- the more intense the colour, the more intense and severe the infection
What does ELISA stand for?
Enzyme linked immunosorbent assay
What are the 2 methods of detecting specific genes?
- probe based methods (labeled, single stranded nucleic acid fragment to hybridize with the target DNA)
- amplification based methods
Culture based methods can be either ___ or ___ media
solid
liquid
What is the culture based method of detecting obligate intracellular organisms?
- very labour intensive and slow
- uses imumunodetection, molecular methods to detect
What do biochemical tests entail?
- they are based on what kinds of things organisms eat - gives you an idea of what kind of organism might be available in the specimen
- these organisms can utilize glucose, lactose, maltose, etc
