Systems for Detection of Pathogens I Flashcards
What is Taxonomy?
the science of naming and classifying organisms
Why is taxonomy important? Why is naming a pathogen insufficient?
Taxonomy allows a better understanding of relationships among species
-a name only implies capacity for pathogenesis and doesn’t include virulence
Clinical importance of taxonomy
Taxonomy is important so we can identify pathogens which are closely related to each other so that tests can be decided on and be very specific rather than have an overall generalisation.
What is a pathogen?
A microbe CAPABLE of causing disease
-don’t have to cause disease
Different types of pathogens
Commensal Non-Pathogen (in host)
Zoonotic Non-Pathogen (in carrier)
Commensal Opportunist (in host)
Commensal Non-Pathogen (in host)
· Present but not capable of causing disease in the host
· E.g. E.coli
· E.g. Bacteroides thetaiotaomicron ‘good bacteria’
Zoonotic Non-Pathogen (in carrier)
· Present but only capable of causing disease in another host
· E.g. E.coli O157:H7 is subclinical cattle (no disease in cattle, but if you eat meat with this toxin you will be ill)
Commensal Opportunist (in host)
· Present and capable of causing disease in the host but only in certain circumstances (e.g. when immunocompromised)
· E.g. Bacteroides fragilis
· E.g. Coagulase Negative Staphylococcus (CNS)
Requirements when sampling a pathogen
Sterile sites must be free from contamination
>E.g. skin flora in blood cultures
Non-sterile sites require decontamination of normal flora
>E.g. faeces, mouth, skin
Samples with high volume or relatively low infected pathogen load require concentration (centrifugation, filtering)
>E.g. CSF, Ascites, 24 hr urine
Do we have to culture organisms to show/detect it is present?
- It is useful to culture the organism to prove its essence, but it is not essential
- It can show that the sample is there and growing by enrichment, purification and amplification of the organism
- Do not need to culture if you already know that the organism is there
Methods used to detect pathogens include…
- Microscopy
- Classical Culture and Identification
- Virology
How is microscopy used to detect pathogens?
Direct Light Microscopy
-used to detect big organisms (bacteria)
Direct Electron Microscopy
- used to detect small organisms (viruses)
- useful in identifying shapes of viruses
Organisms seen under light microscope
Trichomonas vaginalis (STD)
· Swims
· Easy to see- take a vaginal sample which can be seen immediately under the microscope
Schistosoma Mansonii
- Found in kidneys, damaging kidney tubules
Entamoeba Histolytica
Strongyloides (thread worm)
· Easy to detect
· Used to pull the sellotape on the anus and the worm would lay eggs on it. Sellotape then put under microscope
Method of identifying bacterium under light microscope
Gram Staining:
· identify the type of bacteria (gram negative or gram positive)
· look at the shape (spirals, rods, coccus, bacillus) and features on the bacteria
· see whether a bacterium has a capsule or not. A capsule is an important feature because capsulated organisms are more likely to be pathogenic
· see whether bacterium contains any spores
Organisms seen under electron microscope
- Rotavirus (Reovirus) from Faeces
- Rabies (Lyssavirus) from Brain tissue
- Hepatitis B (Hepadnavirus) from Liver
- Tonsillitis (Adenovirus) from Nasal Secretion
How can we identify the presence of a virus under a light microscope?
Immunofluorescent Staining with Pathogen Specific Conjugated Antibody:
· Use antibodies with a fluorescent stain to look for the cell that has been infected by the organism
This technique is used to see the measles virus growing inside the cell. The measles viruses themselves are too small to be seen by light microscopy. The antibody sticks to the virus and therefore the cell fluoresces, which can be seen under the light microscope.
Advantages of using microscopy to identify pathogens
- easy to perform
- rapid screening
- some parasites have SPECIFIC morphology under microscope (e.g. Schistosoma mansonii)
- specific immunofluorescence staining possible
Disadvantages of using microscopy to identify pathogens
-Not sensitive (hence why we culture)- e.g. TB sample required at least 10,000 organisms per ml to be visualised
- General stains are not specific
- Labour intensive, which is expensive
- Requires specialist interpretive expertise, more expensive
Which pathogens can we culture?
Bacteria (not viruses)
How do we culture and identify bacterial pathogens?
Use agar plates with different electrolytes and carbon sources for the bacteria to grow and form colonies
Bacterial culture media
nutrients used to support the growth of bacteria in the laboratory (in vitro)
Medium used in bacterial culture
· Non-Selective Media (e.g. Blood Agar)
· Semi-Selective Media (e.g. MacConkey Agar, DCA, CLED)
· Selective Growth Temperatures (e.g. Campylobacter species)
Conditions required for a bacterial culture
Selective media
Selective atmosphere
Selective temperature
Bacterial Culture: Selective Media
suppress growth of unwanted bacteria and encourage growth of desired pathogens for identification
Examples of selective media
Deoxycholate Agar (DCA) Medium -selective for Shigella and Salmonella on faecal sample
Cysteine Lactose Electrolyte Deficient (CLED) Agar
-selective for non-lactose fermenting Enterobacteria from urine sample
Bacterial Culture: Selective Atmosphere
We can also select the pathogen media depending on what conditions they grow in e.g.:
- aerobic (high O2)
- anaerobic (high CO2)
Anaerobic organisms in culture
Anaerobic organisms will only grow in an atmosphere with no oxygen. This is because they don’t have the capacity to deal with oxygen’s effects on their metabolism. Anaerobic organisms include:
- Clostridium tetani
- Clostridium botulinum
- Clostridium difficile
- Clostridium perfringens
- Bacteroides fragilis
What does frostbite cause?
If you have frostbite, tissue is degrading and there is no oxygen supply. Hence, anaerobic organisms are able to grow there and cause infection (Clostridium perfringens).
- Clostridium perfringens is an aerotolerant anaerobe which produces spores in environmental conditions and exotoxins in humans causing food poisoning and gangrene (necrotic tissue)
How do we detect for gangrene by using culture?
· Tryptose sulphite cycloserine (TSC) agar will show gangrene as black sulphites producing colonies, some with lecithinase (white circles) → grown in anaerobic conditions
Bacterial Culture: Specific Haemolysis of Blood
Some microorganisms will undergo haemolysis. This can be a good indicator of what type of organism they are.
· Particular protein is used to lyse the blood, resulting in either alpha or beta haemolysis
Metabolic Testing in bacterial culture
To see whether an organism causes fermentation e.g. by containing certain enzymes:
- Catalase → E.coli= +ve; Clostridium perfringens= -ve
- Can cleave indole from tryptophan (indole test) → E.coli= +ve; Clostridium perfringens= -ve
Classical systematic bacteriology
We can make a table to show the pattern that a specific pathogen has therefore when conducting the test, it is easy to identify which pathogen it is.
· This is systematic taxonomy: taxonomically identifying the pathogen by observing its biochemical reactions in the lab
Antibiotic Sensitivity Testing
· Use antibiotic discs and identify which organisms are resistant or not. This can only be done if the organism can actually grow
· Also take a strip and show how resistant the organism is to antibiotics as the antibiotics get stronger further along the strip. This is useful to identify which dosage should be used for patients depending on how resistant the organism/pathogen is.
How can we use virology to detect pathogens?
We can’t grow viruses because they are intracellular organisms, meaning they have to use the cellular mechanisms of other host cells to divide.
1) Culture
- requires permissive cell lines e.g. Vero cells (kidney epithelial cells for Herpes Simplex)
- Cytopathic Effect
- Immunofluorescent staining of culture
2) Direct Antigen Detection
- ELISA (e.g. Influenza virus)
- Measles in Vero cells
Direct and Serological ELISA to detect virus
1) Make antibodies for different types of proteins on the surface of viruses
2) Capture the virus and put antibody with a conjugate which has an enzyme on the end of it
3) Then add the chromogenic substrate and if it has the virus it will produce a colour
How is ELISA used in influenza detection?
In influenza there are two major antigens called Haemagglutinin (4 major types) and Neuraminidase (2 major types).
The antibody made in ELISA is either specific for haemagglutinin or neuraminidase and if it is has the specificity it will show a colour. This will tell us the type of proteins and then the type of virus → used to be able to tell which type/strain of influenza virus.
