Diagnosis of Infections Flashcards
What are the goals of a clinical microbiology laboratory?
Identification of the microorganism in the patient specimen involved in the disease process
Provide information on the antimicrobial susceptibility of the isolated microorganism
What are the goals of laboratory tests?
Detection of microorganisms or their products in specimens collected from the patient
Detection of the patient’s immune response to infection
What are the three main categories that laboratory tests for clinical microbiology are divided into?
Identification of microorganisms by isolation and culture
Identification of specific microbial gene or product
Detection of specific antibodies to a pathogen
What does the identification of microorganisms by isolation and culture tell us?
It determines the presence/absence or number or microorganisms and it can be further used to determine susceptibility
These tests are the gold standard
What does the identification of specific microbial genes or products tell us?
The identification of an organism based on the presence or absence of certain cell components, extracellular products, specific gene sequences.
The presence or absence of resistance genes can also be determined (but does not necessarily determine susceptibility)
These tests can yield more rapid results
Why is the detection of specific antibodies to a pathogen important?
The collection of specific IgM or IgG antibodies can be helpful in the acute phase of illness for diagnosis
These tests are important when a pathogen cannot be cultured or when the pathogens pose a high risk for the laboratory workers
These tests tend to result in delayed or retrospective diagnosis
Specimens intended for cultivation of microorganisms can be divided into what two types?
Those from sites that are normally sterile
Those from sites that usually have commensal flora
What is a major problem with culture based methods?
A routine culture takes more than 18 hours minimum to produce a result
What are the advantages of non-culture-based methods?
They are fast, less labour intensive and suitable for organisms that cannot be cultured in the lab
What are three different types of non-culture-based methods?
Microscopy
Immunodiagnostics
Molecular diagnostics
What are the different types of microscopy?
Light microscopy (which includes bright field microscopy, the most commonly used, as well as dark field, phase contrast and fluorescence microscopy) Electron microscopy (transmission EM, scanning EM and immuno EM)
What types of tests are done with light microscopy?
Gram stains (which indicates the morphology and gram reaction) Acid fast (Ziehl-Neelsen Staining) Macromolecule staining (staining cells for their storage pods/granules)
How does a Gram stain work?
Microorganisms are fixed on a slide
Crystal violet dye is added
Iodine is added and it forms a complex with crystal violet
Alcohol removes the iodine-crystal violet complexes from Gram negative organisms (Gram positive organisms retain the complexes via their peptidoglycan)
Pink safranine is added
Gram positive organisms will appear purple and gram negative organisms will appear pink
What is acid fast staining?
Mycobacterium contain a lipid called mycolic acid in their cell wall. When they are stained with fuschin, they withstand decolorization with acid and alcohol
How is fluorescent staining used?
Some organisms are naturally fluorescent and some can be stained with fluorescent dyes and viewed with ultraviolet light.
This is often used to detect the presence of antigens by ‘staining’ samples with antibodies labeled with fluorescent dyes. There is direct and indirect tests (use of a second antibody)
How does an electron microscope work?
It uses electron beams instead of light and magnets instead of lenses
What is one problem/requirement of electron microscopy?
It requires thin specimens because electron beams penetrate poorly. Microbial cells are too thick so they are fixed and mounted in plastic and cut into thin sections
What is the advantage of electron microscopy?
Higher magnification because of resolution (able to identify two object that are very similar looking s two separate objects)
It’s primarily used for viruses
Not routinely used in clinical labs
What is scanning electron microscopy used for?
To look at external structures
What is transmission electron microscopy used for?
To look at internal structures
What is the advantage to immunodetection?
It’s much more rapid than culturing
What are the two main methods of immunodetection?
Those that detect antigens by their interactions with specific antibodies
Those that detect microbial toxins
What is a common method of diagnosing the causative agents of bacterial meningitis? What is the problem with this method?
Specific antibodies can be coated onto latex particles and they will react with the organism or its products, resulting in visible clumping. In the case of bacterial meningitis, the common causative agent (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningiditis) can be detected in the CSF via this method.
The problem with this method is can have false-positives
What is the most common immunodetection test?
Enzyme-linked immunosorbent assay (ELISA)
How does a direct ELISA work?
A 96-well plate is coated with an antibody against a specific antigen.
The patient specimen is added.
If the antigen is present, it will bind to the antibody
Add a second antibody that is linked with an enzyme
Add the substrate for the enzyme
If the antigen is present, the second antibody will bind and the products of the enzyme will be present (causes colour formation; colorimetric).
A negative test won’t have a colour change
How does an indirect ELISA work?
A 96-well plate is coated with an antigen
The patient specimen is added.
If the antibody against that specific antigen is present, it will bind to the antigen
Add a second antibody that is linked with an enzyme
Add the substrate for the enzyme
If the antibody is present, the second antibody will bind to it and the products of the enzyme will be present (causes colour formation; colorimetric).
A negative test won’t have a colour change
ELISA’s can also be used for quantification
What is the main difference between a direct and indirect ELISA?
Direct: you’re looking for the components of the pathogen
Indirect: you’re looking at the response of the patient to the antigen
When running a diagnostics lab, which method is preferable: direct or indirect ELISA?
Indirect because you can use the same secondary antibody for multiple diagnostic tests (it binds to the constant region of the first antibody)
What are hybridomas?
They are produced by the fusion of ‘immortal’ B-cell tumours and individual normal antibody producing cells
What do hydridomas do?
They provide a copious source of monoclonal antibodies, all with identical specificities for their relevant antigen
What are monoclonal antibodies used for?
They are used as diagnostic tools
In direct ELISAs, they are used to detect antigens from patient (detection of rotavirus, HIV, HBV, Herpes, respiratory syncytial virus)
What are two methods for the detection of specific genes?
Probe-based methods
Amplification-base methods
What are probe-based methods?
A gene probe is a nucleic acid molecule that, when in the single-stranded state and labelled, can be used to detect a complementary sequence of DNA by hybridizing to it.
What is important when designing a probe?
You need to keep in mind that he probe needs to be short (the optimum length is generally between 100 and 500 base pairs)
What is PCR?
Polymerase chain reaction
It can amplify a target sequence of DNA from any source, making it easy to identify the organism
How does PCR work?
The DNA strands are heated up (70ºC) so that the hydrogen bonds break and they become single stranded
Primers are added (20-30 base pairs) and they bind to the target gene
Taq polymerase (which can function at high temperatures) will synthesize the daughter strands
PCR cannot be used for quantification
What are different types of growth medium?
Solid or liquid medium
Selective medium
Differential medium
Antimicrobial susceptibility of the medium
What type of solid medium do we use most often? How do we determine growth?
We use agar most often because most bacteria cannot break it down
It allows for the isolation of organisms. You can streak the agar and isolate the colonies and identify the organisms
How is growth determined with liquid medium?
It is based on the turbidity
What is selective medium?
It allows for the growth of certain microorganisms while inhibiting the growth of others
What is differential medium?
It allows the growth of two different organisms but it allows you to differentiate between the organisms
What are the different types of hemolysis?
Alpha hemolysis (incomplete hemolysis which causes the release of heme, creating a green pigmentation) Beta hemolysis (complete hemolysis) Gamma hemolysis (no hemolysis)
How do we culture viruses?
The most common method is by the cytopathic effect
The detection of viruses is based on the change in cellular morphology
What are the problems with cell cultures?
They are labour intensive, slow and easily contaminated
We typically use immunodetection and molecular methods instead
What is the first test done in identifying bacteria?
Gram stain and morphology determination
If we have a gram positive rod, what is the next test?
We determine if it’s anaerobic or aerobic
If it’s anaerobic, it’s likely Clostridium or Actinomyces
If it’s aerobic, it’s likely Cornebacterium, Bacillus or Listeria
If we have a gram positive cocci, what’s the next test?
Determine if it’s Staphylococcus (clusters) or Streptococcus (pairs or chains)
If we determine that we have Staph, what’s the next test?
Determine if it’s coagulate positive (S. aureus) or negative (S. epidermidis or S. saprophyticus)
If we determine that we have Strep, what’s the next test?
Hemolytic test
If we have a gram negative rod, what’s the next test?
Is it anaerobic (Bacteroides, Fusobacterium) or aerobic
For aerobics, you need to determine if they require fastidious growth requirements (Legionella) or simple growth requirements (lactose fermenters, non-lactose fermenters and others)
If we have gram negative cocci, what’s the next test?
There are many, it means it’s likely Neisseria (or Moraxella)
What are biochemical tests?
You can identify microorganisms by phenotypic array. For example, if an organism uses sugar as its carbon source, you can detect its presence by the production of acid
