Systems For Detecting Pathogens I Flashcards
- What are some important features that a pathogens name needs to have
- Needs to be descriptive eg what type of pathogen, what disease it causes
- Uses Systematic bacteriology and virulence
- What do names tell us in terms of pathogenicity or virulence?
Names only imply capacity for pathogenicity
They do not include an assessment of virulence
- What forms the name of the pathogen “Mycobacterium Tuberculosis”
So we can look at taxonomy and see that the domain is a eurobacteria, at the genus level its called a mycobacterium and it causes TB
- What are the three obligate pathogens in humans of the mycobacterial?
Mycobacterium tuberculosis (MTB)
M. leprae
M. ulcerans
- The typical definition of a pathogen is “A biological agent that causes disease or illness to its host” why is this not necessarily right?
what if it was present and not causing disease? Maybe its latent , maybe the patient is a carrier
- Whats a commensal non pathogen (in host) and give an example of one
e.g. E.coli = present but not capable of causing disease in the host
- Whats a Zoonotic Non Pathogen (in carrier) and give an example of one?
present but only capable of causing disease in another host e.g. E.coli 0157:h7 in cattle is subclinical , however if humans gets this E.coli they would be very ill
- What is a commensal opportunistic (host) and give an example?
present and capable of causing disease in the host but only in certain circumstances (immunocompromised) eg Bacteroides Fragilis
- Are all positive samples of a pathogen diagnostic of disease?
being presented with the organism is not always a guarantee that you’re going to get the disease
- What % of humans are infected with Mycobacterium Tuberculosis, what % have the latent disease?
30% of humans are infected : 18% have LATENT disease
- What % of humans are infected with Helicobacter pylori , what % have the latent disease?
50% of humans are infected : 25% have LATENT disease
- How should we define a pathogen?
A microbe CAPABLE of causing a specific degree of host damage
- What controls are important during a test to ensure accurate results?
- Sterile sites must be free from contamination eg. Skin flora in blood cultures
- Non sterile sites require decontamination of normal flora eg Faeces, Mouth, Skin
- Samples with high volume or relatively low infected pathogen load require concentration (centrifugation, filtering) eg CSF, Ascites, 24 hr Urine
- Do samples really need to be cultured?
It depends what you’re looking for
- You don’t really need to, we can amplify the sample for an identification of the organism inside
- Don’t necessarily need to as long as we think it’s there
- BUT you Need to have a rough idea of what you’re looking for
- Explain the difference if you do with culturing or directly?
Culture: Preparation Phase (Enrichment , Amplification and Purification)
Direct: Preparation Phase (Concentration, sample treatment)
Both have the same identification phase
Identification Phase (Molecular DNA/RNA
Gross morphology -Microscopy-
Chemical composition -HPLC MassSpec-)
- How can we detect pathogens using a light microscopy - what kind of pathogens can we detect and why?
- -We can use microscope for very large organisms – Trichomonas Vaginallis ( you can see very easily with a microscope, the most common STD in the world)
- Don’t need to do PCR, culture etc..
- Give some examples of pathogens that are large enough to be detected using light microscopy?
- -Strongyloides (threadworm) very easy to detect with a microscope
- -Entamoeba Histolytica
- -Schistisoma Mansomii
- What can we use electron microscopy for?
- Small organisms
- Looking at different types of shapes might help you understand what you’re looking for however its not diagnostic . Very useful but not usually used in a diagnostic setting
- Give some examples of pathogens we can find using electron microscopy and where in the body would we find them??
- Rotavirus (Reovirus) from Faeces
- Rabies (Lyssavirus) from Brain tissue
- Hepatitis B (Hepadnavirus) from Liver
- Tonsilitis (Adenovirus) from Nasal secretion
- One way of detecting pathogens is by bacterial staining them using light microscopy. how does this work?
You can look at the bacteria for different things eg what gram state they are in, what the organisms look like (for eg do they have a flagella), it has capsules or spores
But you would need to do another diagnostic test to confirm
- Are capsuled organisms more pathogenic or less?
capsulated organisms are more likely to be pathogenic
- Is it easy to see viruses down a microscope?
no, its very difficult to see a virus down a light microscope
- What mechanism can we use to detect viruses?
Immunofluorescent staining with pathogen specific conjugated antibody:
- Take the viruses which are inside cells
- Add antibodies with a fluorescent tag
- Look for cell infected with the organisms
- What are some advantages of microscopy?
Easy to perform Rapid screening Some parasites have SPECIFIC morphology eg. schistosoma mansoni Specific Immunoflourescence staining possible
- What are some disadvantages of microscopy?
Not Sensitive
eg.Mycobacterium Tuberculosis
screening sputum smears requires
at least 10,000 orgs per ml to be visualised
General stains are not specific
Labour intensive (expensive)
Requires specialist interpretive expertise (more expensive)
- Culturing the pathogen relies on what?
This relies on the ability of the test system
to be able to grow the pathogen
- What are three types of media we can use for culturing pathogens - give examples of each?
Non Selective Media
eg. Blood Agar
Semi Selective Media
eg. MacConkey Agar, DCA, CLED
Selective growth temperatures
eg. Campylobacter species
- What is a selective medium?
Selective media = only grow the pathogens – do this by how they grow
- Gangrene is caused by an anaerobic bacteria, how can we use this information to create a selective medium?
Anaerobic area of where the pathogen is – haven’t got oxygen – pathogen has an opportunistic event to grow–> growth = pathogen!
- What is selective atmosphere?
Using the atmosphere of the medium to favour pathogen growth eg the pathogen is only in oxygen or in the lungs (co2) so cater culture to their adaptations or anaerobic atmosphere (they don’t have capacity to deal with what oxygen does )
- What is selective temperature?
Using a temperature that just favours the growth of the pathogen for eg Campylobacter only grows in cows at 42 degrees, so culture should be 42 degrees
- What is the Specific haemolysis of blood?
rupturing (lysis) of red blood cells (erythrocytes) and the release of their contents (cytoplasm) into surrounding fluid
alpha and beta = good indication
- Give an example of a bacteria that grows in :
- Aerobic
- Carbonated
- Aneraboic
conditions. .
O2 = E.c oli CO2 = Haemophilus influenzae ANO2 = Clostridium perfringens
- Whats the Indole test?
Can cleave indole from tryptophan
- What is the Classical systematic bacteriology
You can make a table of pathogens and their different results from tests eg (indole test) so you form patterns with different pathogens
36. On a Antibiotic sensitivity plate testing, what do the following letters stand for? TE E P C VA CTX
TE = Tetracyclin E = Erythromycin P = Penicillin G C = Chloramphenicol VA = Vancomycin CTX = Cefotaxime
- How would you determine whats causing food poisoning in a patient?
- Take a sample of stool
- Either :
-> Direct microscopy for cysts/eggs of
amoebae or parasites
- > incubate aerobically at 37ºC–> culture on macconkey agar –>dentify bacteria by biochemical profiling (API 20E strip)—> Could either be Salmonella typhimurium OR Shigella flexneri –> Antibiotic sensitivity testing disc diffusion plates
- > incubate microaerophilically at 42ºC–> Spread on Campy CVA selective media plate–> Positive growth (silver colonies)–>Gram stain–> shows gram negative curved rods–> if its oxidase positive = Campylobacter jejuni
- Why cant we culture viruses the same way we culture bacteria?
We cant grow viruses because they have intracellular
Viruses are defined that they NEED hosts cells
- How can we grow/culture viruses?
* we dont usually do this anymore**
Culture and Microscopy?
Requires cell lines (eg vegro cells ) saw cytopathic effects of CMV and HHV-1
Takes forever and difficult. But only real way to grow viruses
shows?
Cytopathic Effect
Immunofluorescent staining of culture
- Aside from “Culture and Microscopy” how else can we see viruses?
Direct Antigen Detection
-ELISA test eg for influenza virus
- How can we use ELISA to detect influenza virus?
We use ELISA – look at under the microscope- can see RNA inside and two major proteins on the outside H and N
Each type on the outside we can make antibody with conjugate with an fluroscent enzyme
Antibody is specific for H and N if its got that specificity it will give you a colour
A MULTIPLE SAMPLE or dilution method (positive and negative and dilute it down) get titre
Titres tell us how strong the virus is
Virus (with H or N antibody)
H or N conjugate
Indicator Enzyme
Substrate
- Compare the following ways to detect infuenza:
-Electron Microscopy
-Culture
-ELISA
?
Electron microscopy can see this virus
but not identify it as ‘swine flu’
Culture takes 3-10 days
Rapid ELISA for fluA antigen = 15 mins
ELISA for Flu Antibody
- What are some advantages of Classical culture and identification?
Cheap simple, reliable reagents
Sensitive
eg. Single organisms can be grown and identified
Validated specificity
eg. ‘Gold Standards’ with multiple parameters
Direct in vivo measurement of effectiveness of therapy
eg Antibiotic sensitivity
Easily archived
eg. Epidemiology
- What are some disadvantages of Classical culture and identification?
Some pathogens cannot be grown eg.Mycobacterum M.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
eg. Mycobacterum Tuberculosis
Labour intensive (expensive)
Requires specialist interpretive expertise (more expensive)
