Diagnosis of Infection

Question 1

What are the 2 ways that a pathogen can be identified?

Answer 1

• 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

Question 2

Which gene can be isolated that is known to cause methicillin resistance?

Answer 2

MECAG gene

Question 3

What two pieces of information is important when identifying a microorganism?

Answer 3

• presence of that microorganism

- susceptibility and the presence of a resistance gene

Question 4

In what cases is it most useful to detect the specific antibodies to a pathogen rather than the actual pathogen?

Answer 4

• pathogens that cannot be cultivated/ takes a long time to cultivate (tuberculosis is mostly identified via presence
• high risk group pathogens
• retrospective diagnosis

Question 5

How does PCR work?

Answer 5

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

Question 6

What are the advantages of non-culture methods?

Answer 6

• fast
• less labor intensive
• suitable for organisms that cannot be cultured in the lab

Question 7

What are some examples of non-culture methods?

Answer 7

• microscopy
• immunodiagnostics
• molecular diagnostics

Question 8

What is the main disadvantage of using an electron microscope?

Answer 8

• cannot image live cells

Question 9

What is resolution?

Answer 9

• the minimum distance that you can distinguish two objects as two objects

Question 10

What are the four steps of the gram stain?

Answer 10

1. flood with crystal violet
2. flood with iodine
3. flood with ethanol
4. flood with safarin

Question 11

What piece of information does the gram stain give you?

Answer 11

• 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)

Question 12

Acid fast cells are ______

Answer 12

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

Question 13

Describe florescent staining?

Answer 13

• 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

Question 14

Describe electron microscopy

Answer 14

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

Question 15

In what cases is imumunodetection used?

Answer 15

• 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

Question 16

What does ELISA allow you to do?

Answer 16

• 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

Question 17

How do you quantify ELISA?

Answer 17

• 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

Question 18

What is the process of using ELISA?

Answer 18

• 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

Question 19

What is ELISA quantified with?

Answer 19

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

Question 20

What does ELISA stand for?

Answer 20

Enzyme linked immunosorbent assay

Question 21

What are the 2 methods of detecting specific genes?

Answer 21

• probe based methods (labeled, single stranded nucleic acid fragment to hybridize with the target DNA)
• amplification based methods

Question 22

Culture based methods can be either ___ or ___ media

Answer 22

solid

liquid

Question 23

What is the culture based method of detecting obligate intracellular organisms?

Answer 23

• very labour intensive and slow

- uses imumunodetection, molecular methods to detect

Question 24

What do biochemical tests entail?

Answer 24

• 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