42. Systems for Detection of Pathogens I

Question 1

How can we define a pathogen?

Answer 1

A microbe CAPABLE of causing a specific degree of host damage

Question 2

How do you get a good sample?

Answer 2

• 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

Question 3

What happens in the preparation phase and in the identification phase when there is a direct sample and a culture?

Answer 3

•PREPARATION PHASE

• Culture = Enrichment Purification Amplification
• Direct = Concentration Sample treatment

•IDENTIFICATION PHASE
- Culture = Molecular DNA/RNA  Gross morphology (Microscopy)
Chemical composition (HPLC MassSpec)
- Direct = Molecular DNA/RNA  Gross morphology (Microscopy)
Chemical composition (HPLC MassSpec)

Question 4

Advantages and disadvantages of microscopy

Answer 4

+ Easy to perform
+ Rapid screening
+ Some parasites have SPECIFIC morphology
+ Specific immunofluorescence staining possible

• Not sensitive ~ e.g. TB
• 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 5

Classical culture and identification ~ bacteriology and examples of the different types of media

Answer 5

• Bacteriology - This relies on the ability of the test system to be able to grow the pathogen
• Environment - O2, CO2, ANO2,
• Quantification, identification, antibiogram

•colony morphology, colour, haemolysis, colony count, colony identification, systematic identification, colony resistance to antibiotics

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

Question 6

What are classical metabolic testings?

Answer 6

•Catalase
E.coli = +ve
Clostridium perfringens –ve

•Can cleave indole from tryptophan (Indole test)
E.coli = +ve
Clostridium perfringens –ve

Question 7

What are classical culture identification - VIROLOGY?

Answer 7

1) Culture & microscopy
- Requires permissive cell lines e.g. vero cells (kidney epithelial) for measles (Morbillivirus)
- Cytopathic effect
- Immunofluorescent staining of culture

2) Direct antigen detection
- ELISA e.g. influenza virus

Question 8

Advantages and disadvantages of classical culture and identification.

Answer 8

+ cheap simple, reliable reagents
+ sensitive e.g. single organisms can be grown and identified
+ validated specificity e.g. gold standards
+ direct in vivo measurement of effectiveness of therapy e.g. antibiotic sensitivity
+ easily archived e.g. epidemiology

• some pathogens cannot be grown
• some p cannot be well differentiated by biochemistry alone
• slow = culture requires at least overnight incubation ~ viral = 3-10days, mycobacterial = 6-12 weeks
• some pathogens grow too slowly to aid rapid diagnosis e.g. TB
• Labour intensive (expensive)
• Requires specialist interpretive expertise (more expensive)