Medical Microbiology: Systems for Detection of Pathogens I

Question 1

What is taxonomy?

Answer 1

• The naming, defining and classiftying of organisms

Question 2

What can the name of a pathogen imply about it?

Answer 2

• Names only imply capacity for pathogenicity
• They do not include an assessment of virulence

Question 3

How can you define what a pathogen is?

Answer 3

• A pathogen is biological agent that causes disease or illness to its host

Question 4

What is the problem with defining a pathogen as something that definitely causes disease?

Answer 4

• Some pathogens may be latent or not at a high enough concentration to cause disease

Question 5

What would a more accurate way of defining a pathogen be?

Answer 5

• A microbe that is CAPABLE of causing a specific degree of host damage

Question 6

What is a commensal non-pathogen?

Answer 6

• A pathogen that’s present but not capable of causing disease in the host
• E.g. E.coli

Question 7

What is a zoonotic non-pathogen?

Answer 7

• A pathogen that’s present but only capable of causing disease in another host
• E.g. E.coli O157:H7 is subclinical in cattle

Question 8

What is a commensal opportunist?

Answer 8

• A pathogen that’s present and capable of causing disease in the host but only in certain circumstances
• E.g. Bacteroides fragilis and Coagulase Negative Staphylococcus (CNS)

Question 9

Are all positive samples diagnostic of active disease, why is this?

Answer 9

• No
• This is because the body’s immune system may fight off the pathogen before it’s able to cause disease
• Also, some pathogens can become latent within the body and cause disease later
• You may not have been exposed to a large enough amout of pathogen for it to cause disease

Question 10

When taking a sample of a pathogen from a patient what things must be done to ensure the sample is good?

Answer 10

• 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 11

Do you need to culture all samples of a pathogen?

Answer 11

• No, you don’t always have to culture an organism/pathogen to prove that it’s there
• This is especially true if you have an idea of what organisms may be present within a particualr sample

Question 12

What is light microscopy used for when identifying organisms in samples?

Answer 12

• Light microscopy used to visualise large organisms

Question 13

What is electron microscopy used for when identifying organsisms in samples?

Answer 13

• Electron microscopy is used to visualise really small organisms, mainly viruses
• Not usually used in diagnostic setting

Question 14

Why would you use electron microscopy to visualise an organism when there more modern techniques that can be used?

Answer 14

• Organism/pathogen can’t be cultured
• PCR for oragnsim hasn’t be defined yet
• Unable to identify what disease a patient has

Question 15

Bacterial staining can be used in conjunction with light microscopy when identifying organisms in samples. What can bacterial staining be used for?

Answer 15

• Used to:
  
  • Identify whether a bcateria is gram negative or gram positive
  • Identify shape of bacteria
  • Idenify presence of spores or capsules
  • Identify bacterial species

Question 16

How can you view viruses using light microscopy?

Answer 16

• Add specific antibody to the viral antigen tagged to a fluorochrome to a sample and then view that sample under microscope
• Antibody will bind to cells with viral antigens so those cells will fluoresece which can be seen on light microscope
• This is called immunofluoresence staining

Question 17

What are the advantages of microscopy?

Answer 17

• Easy to perform
• Rapid screening
• Some parasites have specific morphology to look out for E.g. schistosome mansonii
• Specific immunofluorescence staining possible

Question 18

What are the disadvantages of microscopy?

Answer 18

• Not very sensitive - screening of sputum smears needs A LOT of organisms to be visualised E.g. mycobacterium tuberculosis
• General stains are not specific
• Labour intensive - expensive
• Needs specialist interpretive expertise - even more expensive

Question 19

What is classical culture?

Answer 19

• Technique that involves use of agar plates with either blood or different types of electrolytes in it as well as some sort of carbon source which allows bactria to grow and form colonies

Question 20

What is classical culture dependent on?

Answer 20

• Dependent on the ability of the test system to be able to grow the pathogen

Question 21

What are the different types of growth medium used in culture?

Answer 21

• Non-selective media e.g. blood agar
• Semi selective media e.g. MacConkey Agar, DCA, CLED
• Selective growth temperatures e.g. Campylobacter species

Question 22

What is a selective medium?

Answer 22

• Growth medium that selects and only grows pathogens

Question 23

What is a selective atmosphere medium?

Answer 23

• Growth medium that’s used to grow pathogens in specific conditions, usually representative of conditions they would usually grow in
• E.g. Aerobic atmosphere; CO₂ rich culture; Microaerophilic Culture - low oxygen levels and Anaerobic culture

Question 24

You can use classical culture to create a system and conduct a large variety of tests on the bacteria within that system to find out which environment it grows best in. What are some of the tests that can be conducted in this situation?

Answer 24

• Quantification, Identification, Antibiogram test E.g.
  
  • Colony morphology/colour
  • Haemolysis
  • Colony Count
  • Colony Identification
  • Systematic identification
  • Colony Resistance to antibiotics

Question 25

Metabolic testing can also be conducted in this bacteria culture system, what are some examples of metabolic tests that can be done?

Answer 25

* **Catalase test** * E.coli = +ve * Clostridium perfringens –ve * **Indole test** - Can cleave indole from tryptophan * E.coli = +ve * Clostridium perfringens –ve

Question 26

How can you identify Enterobacteriacae?

Answer 26

* Metabolic function and sugar utilisation tests

Question 27

What is antibiotic sensitivity testing?

Answer 27

* Test used to identify which antibiotics particular bacteria are sensitive/insensitive to * Allows for an antibiotic profile to be made for a particular bacteria * Can also be sued to find out how much of an antibiotic is needed to kill bacteria (E.test)

Question 28

Why can't viruses be grown using culture?

Answer 28

* Viruses are intracellular oragsnisms and so require cellular mechanisms of the host cell to divide

Question 29

How can viruses be grown?

Answer 29

* Can be grown using permissive cell lines e.g. Vero cells (kideny epithelial) for herpes simplex virus

Question 30

How can viruses be identified when growing them using permissive cell lines?

Answer 30

* Can be identified via cytopathic effect they produce, changes in the way the cells they infect grow

Question 31

What are the problems with using the cytopathic effect to detect viruses?

Answer 31

* Takes a long time and is difficult to carry out

Question 32

What technique is used to identify viruses instead of ciewing cytopathic effect in permissive cell lines?

Answer 32

* Antigen detection via direct ELISA or antibdoy detection via serological ELISA

Question 33

What are the advantages of classical culture?

Answer 33

* **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 measurement of effectiveness of therapy** e.g. Antibiotic sensitivity * **Easily archived** e.g. Epidemiology

Question 34

What are the disdvantages of classical culture?

Answer 34

* **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