Systems for Detecting Pathogens I Flashcards
Why is it important to name a pathogen?
- Names provide us with the opportunities to define boundaries.
- It is up to the test system to define these boundaries and provide a measure that inform us “ to what extent can any one microbe be able to cause disease”.
What is taxonomy? Why is it important to use taxonomy?
Taxonomy is important to know which organisms are close so that tests can be decided on and be specific rather than using generalisations.
Mycobacterial genous
- Look at the taxonomy and see every single bacterial species that exists
- Currently 148 species
- Only three are obligate human pathogens: M. tuberculosis, M. leprae, M. ulcerans.
Define a pathogen
A microbe capable of causing a specific degree of host damage.
Commensal Non-pathogen (In host)
Present but not capable of causing disease in its host e.g. good bacteria such as E. coli, bacteroides thetaiotamicron
Zoonotic Non-pathogen (in carrier)
Present but only capable of causing disease in another host e.g. E. coli O157:H7 is subclinical in cattle but in humans can cause you to be very ill when eaten.
Commensal Opportunist (in host)
Present and capable of causing disease in the host but only in certain circumstances e.g. Bacteroides fagilis. If the patient becomes immunocompromised, then these pathogens may become harmful.
Why are some positive samples not diagnostic of disease?
Some people can be exposed to an infection however only about 30% will be infected and the other 70% will not be infected. Of those infected, only 40% will have the active infection and the other 60% will have a latent infection. Only 10% of the latent patients will have the active version later. TB is an example
Different samples and how it can affect test results
- Samples from a sterile sites must be free from contamination to prove their is no pathogens e.g. skin flora in blood cultures. If not sterile, then it has pathogens that can cause disease.
- Non-sterile sites require decontamination of the normal flora e.g. faeces, mouth, skin. Only select the pathogens to see the cells that have the potential to cause disease.
- Samples with high volume or relatively low infected pathogen load require concentration (centrifugation, filtering) e.g. CSF, Ascites, 24hr urine
Is culturing a necessity?
- Culturing is only used to show that a organism is present. It is not essential.
- Can enrich, purify and amplify for identification.
- Will need to use a specific test to identify for a desired organism
- Do not need to culture if you already know the organism is there.
Microscopy usage
- Useful
- Cheap
- Simple
- Used for large and small things
What are direct light microscopies used for? Give examples of organisms
Big samples
- Trichomonas vaginalis
- Schistosoma mansonii
- Entamoeba Histolytica
- Strongyloides
Trichomonas Vaginalis under light microscopy
- Sexually transmitted disease
- Swims
- Most common in the world
- Easy to see - take a vaginal sample on the microscopy and can be seen straight away.
- 160 million people infected
Schistosoma mansonii under light microscopy
Found in the kidneys
- 83 million people infected
Entamoeba Histolytica
- 50 million people affected
Strongyloides (thread worm)
- 50% UK children
- Easy to detect
- Used to put the sellotape on the anus and the worm would lay eggs on it, then this can be put under a microscope
What are direct electron microscopy used for? Give examples of pathogens
Little Samples that may not be able to be PCR, cultured or not classified yet
- Rotavirus (Reovirus) from faeces
- Rabies (Lyssavirus) from brain tissue
- Hepatitis B (Hepadnavirus) from Liver
- Tonsilitis (Adenovirus) from Nasal secretion
Electron microscopy is useful but not usually used in a diagnostic setting.
Why is direct bacterial staining used?
It is used to look at the shapes (rod, coccus, bacillus) and features on the bacteria.
Bacillus anthracis
Has a distinct capsule like streptococcus pneumoniae. No other organism has it’s shape so can use a microscopy alone to identify it.
Immunofluorescent staining with pathogen specific conjugated antibody
- Take viruses from inside the cells
- Use antibodies with a fluorescent stain on the outside to look for the cell that has been affected by the organism.
The microscopy is important to detect bacteria and viruses
Measles and immunofluorescent staining
- See the measles virus growing inside a cell.
- The measle viruses themselves are too small to be seen by a light microscopy.
- The antibody which is stuck to the virus and making the cell light up in front of the microscopy.
Advantages of microscopy
- Easy to perform
- Rapid screening
- Some parasites have SPECIFIC morpholgy e.g. schistosoma mansonii
- Specific immunofluorescene staining possible
Disadvantages of microscopy
- Not sensitive e.g. Mycobacterium tuberculosis screening sputum smears require at least 10,000 orgs per ml to be visualised
- > Reason why culturing and PCR is preferred is because the majority of patients will not have that many organisms in their sample.
- General stains are not specific
- Labour intensive (Expensive)
- Requires specialist interpretive expertise (more expensive)
Classical culturing
- Usual way of culturing is using agar plates with different electrolytes and carbon source for the bacteria to grow and form colonies.
- Can see if the culture is pure - for example useful to see if a patient has a urinary tract information
What does bacteriology rely on?
This relies on the ability of the test system to be able to grow the pathogen.
What is needed in the test system to grow the pathogen?
- Non-selective media: e.g. blood agar
- Semi-selective media: MacConkey Agar, DCA, CLED
- Selective growth temperatures: e.g. Campylobacter species
Why is selective media used?
Used to only grow the desired pathogens for example:
- Non-lactose fermenting: Enterobacteria growing on Cysteine Lactose Electrolyte Deficient (CLED) Agar from a urine sample.
- Deoxycholate Agar (DCA) Medium Selective for Shigella and Salmonella on a faecal sample.
Only the pathogens will grow and the non-essentials won’t. Also, select the pathogen media depending on what conditions they grow in, for example; aerobic, anaerobic etc.
Respiratory pathogens
- Neisseria meningitidis
- Neisseria gonorrhoeae
- Haemophilus influenzae
- Brucella melitensis
Anaerobic cultures
Organisms will only grow in an atmosphere with no oxygen. For example, in frostbite, where some tissue is degrading only anaerobes will grow..
What do aerotolerant anaerobes produce?
they produce spores in environmental conditions and exotoxins in humans causing food poisoning and gas gangrene.
Gangrene
The growth of an anaerobic organism gorwing where an area of tissue does not have oxygen. e.g. Tryptose sulphite cycloserine (TSC) agar will show black sulphide producing colonies.
What organisms have a selective temperature and selective atmosphere?
Campylobacter on cows - 42 degrees cows
Why is haemolysis of blood used?
Use this to look at how an organism occurs and identify what type of organism they are.
- Alpha and beta haemolysis - what protein they use to lysis the blood
Classical Metabolic Testing
Use metabolic testing to see whether an organism causes things to ferment
- > Catalase: E. coli = +ve; Clostridium perfringens = -ve
- > Can cleave indole from tryptophn (indole test): E.coli = +ve; Clostridium perfringens = -ve
Classical systematic bacteriology
Systematic taxnomy is 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. Conducted since the 1940s.
What is a phage type?
Organisms particulary bacteria are susceptible to bacteriophages. They are only found in these organisms therefore, some systems are used to phage type organisms to identify what they are.
Describe the 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 - dependent on fast growing organisms e.g. overnight.
- 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.
Why is antibiotic sensitivity testing used?
All of this testing can be used to identify which organism is causing the disease for example in food poisoning - difficult as all organisms produce mostly the same symptoms.
What testing mechanisms are used for virology?
- Culture and Microscopy - not done anymore
a) Requires permissive cell lines to be able to grow the virus e.g. vero cells (kidney epithelial) for Herpes simpelx
b) Cytopathic Effect
c) Immunofluorescent staining of culture - Direct Antigen Detection -
a) ELISA e.g. influenza virus
b) Measles in Vero cells
What is ELISA?
An electron microscopy is used to see the virus but not identify it as “swine flu” for example:
- The culture takes 3-10 days.
- Rapid ELISA for fluA antigen = 15 mins
- ELISA for Flu Antibody.
What are the two proteins on the outside of the influenza virus?
Haemagglutinin
Neuraminidase
ELISA and Influenza Virus
- Make antibodies against these different types of proteins on the outside of the virus.
- Capture the virus and put antibody with the conjugate.
- This has an enzyme on the end.
- Then add the substrate and if it has the virus will make a colour.
The antibody is either specific for haemaglutinin or neuraminidase and if it has the specificity it will show a colour.
ELISA antigens
ELISA titres to specifc antigens can be measured by:
- > Multiple samples single antigen with signal positive colour cut off.
- > Multiple antigens but serial dilution of sample
What is a titre?
Dilution of sample that gives a signal equal or greater than the positive control cut off.
Advantages of classical systems
- Cheap, simple, reliable reagents
- Sensitive e.g. single organisms can be grown and identified
- Validated specificity e.g. ‘Gold Standards’ with multiple parameters
- Direct in vivo measurements
- Easily archived e.g. Epidermiology
Disadvantages of classical systems
- Some pathogens cannot be grown e.g. Mycobacterium leprae
- Some pathogens cannot be well differentiated by biochemistry alone
- Slow: culture requires at least overnight incubation:
- > Viral: 3- 10 days
- > Mycobacterial = 6 - 12 weeks
- Some pathogens grow too slowly to aid rapid diagnosis e.g. Mycobacterium tuberculosis.
- Labour intensive (expensive)
- Requires specialist interpretive expertise (more expensive)
