Systems for Detection of Pathogens I

Question 1

What is Taxonomy?

Answer 1

the science of naming and classifying organisms

Question 2

Why is taxonomy important? Why is naming a pathogen insufficient?

Answer 2

Taxonomy allows a better understanding of relationships among species
-a name only implies capacity for pathogenesis and doesn’t include virulence

Question 3

Clinical importance of taxonomy

Answer 3

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.

Question 4

What is a pathogen?

Answer 4

A microbe CAPABLE of causing disease

-don’t have to cause disease

Question 5

Different types of pathogens

Answer 5

Commensal Non-Pathogen (in host)

Zoonotic Non-Pathogen (in carrier)

Commensal Opportunist (in host)

Question 6

Commensal Non-Pathogen (in host)

Answer 6

· Present but not capable of causing disease in the host

· E.g. E.coli
· E.g. Bacteroides thetaiotaomicron ‘good bacteria’

Question 7

Zoonotic Non-Pathogen (in carrier)

Answer 7

· 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)

Question 8

Commensal Opportunist (in host)

Answer 8

· 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)

Question 9

Requirements when sampling a pathogen

Answer 9

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

Question 10

Do we have to culture organisms to show/detect it is present?

Answer 10

• 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

Question 11

Methods used to detect pathogens include…

Answer 11

• Microscopy
• Classical Culture and Identification
• Virology

Question 12

How is microscopy used to detect pathogens?

Answer 12

Direct Light Microscopy
-used to detect big organisms (bacteria)

Direct Electron Microscopy

• used to detect small organisms (viruses)
• useful in identifying shapes of viruses

Question 13

Organisms seen under light microscope

Answer 13

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

Question 14

Method of identifying bacterium under light microscope

Answer 14

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

Question 15

Organisms seen under electron microscope

Answer 15

• Rotavirus (Reovirus) from Faeces
• Rabies (Lyssavirus) from Brain tissue
• Hepatitis B (Hepadnavirus) from Liver
• Tonsillitis (Adenovirus) from Nasal Secretion

Question 16

How can we identify the presence of a virus under a light microscope?

Answer 16

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.

Question 17

Advantages of using microscopy to identify pathogens

Answer 17

• easy to perform
• rapid screening
• some parasites have SPECIFIC morphology under microscope (e.g. Schistosoma mansonii)
• specific immunofluorescence staining possible

Question 18

Disadvantages of using microscopy to identify pathogens

Answer 18

-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

Question 19

Which pathogens can we culture?

Answer 19

Bacteria (not viruses)

Question 20

How do we culture and identify bacterial pathogens?

Answer 20

Use agar plates with different electrolytes and carbon sources for the bacteria to grow and form colonies

Question 21

Bacterial culture media

Answer 21

nutrients used to support the growth of bacteria in the laboratory (in vitro)

Question 22

Medium used in bacterial culture

Answer 22

· Non-Selective Media (e.g. Blood Agar)
· Semi-Selective Media (e.g. MacConkey Agar, DCA, CLED)
· Selective Growth Temperatures (e.g. Campylobacter species)

Question 23

Conditions required for a bacterial culture

Answer 23

Selective media
Selective atmosphere
Selective temperature

Question 24

Bacterial Culture: Selective Media

Answer 24

suppress growth of unwanted bacteria and encourage growth of desired pathogens for identification

Question 25

Examples of selective media

Answer 25

``` 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

Question 26

Bacterial Culture: Selective Atmosphere

Answer 26

We can also select the pathogen media depending on what conditions they grow in e.g.: - aerobic (high O2) - anaerobic (high CO2)

Question 27

Anaerobic organisms in culture

Answer 27

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

Question 28

What does frostbite cause?

Answer 28

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)

Question 29

How do we detect for gangrene by using culture?

Answer 29

· Tryptose sulphite cycloserine (TSC) agar will show gangrene as black sulphites producing colonies, some with lecithinase (white circles) → grown in anaerobic conditions

Question 30

Bacterial Culture: Specific Haemolysis of Blood

Answer 30

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

Question 31

Metabolic Testing in bacterial culture

Answer 31

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

Question 32

Classical systematic bacteriology

Answer 32

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

Question 33

Antibiotic Sensitivity Testing

Answer 33

· 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.

Question 34

How can we use virology to detect pathogens?

Answer 34

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

Question 35

Direct and Serological ELISA to detect virus

Answer 35

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

Question 36

How is ELISA used in influenza detection?

Answer 36

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.