Systems for Pathogens 1

Question 1

What is in a name

Answer 1

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 informs us

Question 2

What is a pathogen

Answer 2

A microbe CAPABLE of causing a specific degree of host damage

Question 3

How can we define a pathogen

Answer 3

Commensal Non pathogen (in host)

• PRESENT but NOT CAPABLE of causing disease in the host - good bacteria

Zoonotic Non pathogen (in carrier)

• PRESENT but only CAPABLE of causing disease in ANOTHER host

Commensal Opportunist (in host)

• PRESENT and CAPABLE of causing disease in the host but only in certain circumstances

Question 4

Are all positive samples, diagnostic of disease

Answer 4

No,
We only get the infection once the pathogen is active, not when it is latent

Question 5

How should you take samples for testing from

Answer 5

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)

Question 6

What happens in the sample preparation phase

Answer 6

Culture:

• Enrichment
• Purifacation
• Amplification

Direct:

• Concentration
• Sample treatment

Question 7

How do we use the samples for analysis

Answer 7

• Molecular DNA/RNA
• Gross morphology (Microscopy)
• Chemical composition (HPLC MassSpec)

Question 8

Why do we use light microscopy for analysis

Answer 8

Used to visualise bigger samples

• Trichomonas vaginalis
• Schistosoma mansonii
• Entamoeba histolytica
• Strongyloides (thread worm)

Question 9

Why do we use electron microscopy for analysis

Answer 9

Used to visualise smaller samples such as viruses

Question 10

Why do we use Direct bacterial staining of Sample

Answer 10

• Used to differentiate what type of bacteria by their cell wall type
• Change the needed antibiotic for that infection
• Visualise other elements such as capsids

Question 11

Why do we use Immunofluorescent staining for analysis

Answer 11

Able to identify specific antigens present using immunofluorescent tagging

Question 12

What are the advantages in using microscopy

Answer 12

• Easy to perform
• Rapid screening
• Some parasites have SPECIFIC morphology
• Specific Immunoflourescence staining possible

Question 13

What are the disadvantages in using microscopy

Answer 13

Not Sensitive, 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)

Question 14

What media are used to grow bacteria

Answer 14

• Non Selective Media
  eg. Blood Agar
• Semi Selective Media
  eg. MacConkey Agar, DCA, CLED
• Selective growth temperatures eg. Campylobacter species

Question 15

In what ways can we select for the atmosphere type the culture is grown in

Answer 15

• Aerobic culture
• Microaerophilic Culture
• Anaerobic culture

Question 16

How can wee use a combination of selection to grow a culture

Answer 16

Selective temperature and Selective atmosphere

• Campylobacter grows at 42°C and at 10% CO₂

Question 17

How is classical metabolic testing used

Answer 17

Differentiates what metabolic pathways exist in a culture

• Catalase
• Indole test

Question 18

How can we systematically classify bacteria

Answer 18

Each organism has specific requirements to grow, when we isolate each condition we can identify what it is

Question 19

How does viral cell cultures work

Answer 19

Grow cells in culture and allow virus to infect it

When virus exerts its cytopathic effect and bursts out of cells we can identify it

Question 20

How can we identify viruses

Answer 20

Culture & microscopy

• Requires permissive cell lines, eg.Vero cells (Kidney epithelial)
  for Measles (M orbillivir
  us)
• Cytopathic Effect Immunofluorescent staining of culture

Direct Antigen Detection

• ELISA eg. Influenza Virus

Question 21

What are the advantages of identification with cell culture

Answer 21

• Cheap simple, reliable reagents
• Sensitive
• Validated specificity
• Direct in vivo measurement of effectiveness of therapy
• Easily archived

Question 22

What are the disadvantages of identification with cell culture

Answer 22

• Some pathogens cannot be grown
• Some pathogens cannot be well differentiated by biochemistry alone
• Slow: culture requires at least overnight incubation
• Some pathogens grow too slowly to aid rapid diagnosis
• Labour intensive (expensive)
• Requires specialist interpretive expertise (more expensive)