Systems For Detecting Pathogens I

Question 1

1. What are some important features that a pathogens name needs to have

Answer 1

• Needs to be descriptive eg what type of pathogen, what disease it causes
• Uses Systematic bacteriology and virulence

Question 2

2. What do names tell us in terms of pathogenicity or virulence?

Answer 2

Names only imply capacity for pathogenicity

They do not include an assessment of virulence

Question 3

3. What forms the name of the pathogen “Mycobacterium Tuberculosis”

Answer 3

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

Question 4

4. What are the three obligate pathogens in humans of the mycobacterial?

Answer 4

Mycobacterium tuberculosis (MTB)
M. leprae
M. ulcerans

Question 5

5. The typical definition of a pathogen is “A biological agent that causes disease or illness to its host” why is this not necessarily right?

Answer 5

what if it was present and not causing disease? Maybe its latent , maybe the patient is a carrier

Question 6

6. Whats a commensal non pathogen (in host) and give an example of one

Answer 6

e.g. E.coli = present but not capable of causing disease in the host

Question 7

7. Whats a Zoonotic Non Pathogen (in carrier) and give an example of one?

Answer 7

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

Question 8

8. What is a commensal opportunistic (host) and give an example?

Answer 8

present and capable of causing disease in the host but only in certain circumstances (immunocompromised) eg Bacteroides Fragilis

Question 9

9. Are all positive samples of a pathogen diagnostic of disease?

Answer 9

being presented with the organism is not always a guarantee that you’re going to get the disease

Question 10

10. What % of humans are infected with Mycobacterium Tuberculosis, what % have the latent disease?

Answer 10

30% of humans are infected : 18% have LATENT disease

Question 11

11. What % of humans are infected with Helicobacter pylori , what % have the latent disease?

Answer 11

50% of humans are infected : 25% have LATENT disease

Question 12

12. How should we define a pathogen?

Answer 12

A microbe CAPABLE of causing a specific degree of host damage

Question 13

13. What controls are important during a test to ensure accurate results?

Answer 13

• 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 14

14. Do samples really need to be cultured?

Answer 14

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

Question 15

15. Explain the difference if you do with culturing or directly?

Answer 15

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-)

Question 16

16. How can we detect pathogens using a light microscopy - what kind of pathogens can we detect and why?

Answer 16

• -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..

Question 17

17. Give some examples of pathogens that are large enough to be detected using light microscopy?

Answer 17

• -Strongyloides (threadworm) very easy to detect with a microscope
• -Entamoeba Histolytica
• -Schistisoma Mansomii

Question 18

18. What can we use electron microscopy for?

Answer 18

• 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

Question 19

19. Give some examples of pathogens we can find using electron microscopy and where in the body would we find them??

Answer 19

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

Question 20

20. One way of detecting pathogens is by bacterial staining them using light microscopy. how does this work?

Answer 20

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

Question 21

21. Are capsuled organisms more pathogenic or less?

Answer 21

capsulated organisms are more likely to be pathogenic

Question 22

22. Is it easy to see viruses down a microscope?

Answer 22

no, its very difficult to see a virus down a light microscope

Question 23

23. What mechanism can we use to detect viruses?

Answer 23

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

Question 24

24. What are some advantages of microscopy?

Answer 24

Easy to perform
Rapid screening
Some parasites have SPECIFIC morphology
eg. schistosoma mansoni
Specific Immunoflourescence staining possible

Question 25

25. What are some disadvantages of microscopy?

Answer 25

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)

Question 26

26. Culturing the pathogen relies on what?

Answer 26

This relies on the ability of the test system | to be able to grow the pathogen

Question 27

27. What are three types of media we can use for culturing pathogens - give examples of each?

Answer 27

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

Question 28

28. What is a selective medium?

Answer 28

Selective media = only grow the pathogens – do this by how they grow

Question 29

29. Gangrene is caused by an anaerobic bacteria, how can we use this information to create a selective medium?

Answer 29

Anaerobic area of where the pathogen is – haven’t got oxygen – pathogen has an opportunistic event to grow--> growth = pathogen!

Question 30

30. What is selective atmosphere?

Answer 30

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 )

Question 31

31. What is selective temperature?

Answer 31

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

Question 32

32. What is the Specific haemolysis of blood?

Answer 32

rupturing (lysis) of red blood cells (erythrocytes) and the release of their contents (cytoplasm) into surrounding fluid alpha and beta = good indication

Question 33

33. Give an example of a bacteria that grows in : - Aerobic - Carbonated - Aneraboic conditions. .

Answer 33

``` O2 = E.c oli CO2 = Haemophilus influenzae ANO2 = Clostridium perfringens ```

Question 34

34. Whats the Indole test?

Answer 34

Can cleave indole from tryptophan

Question 35

35. What is the Classical systematic bacteriology

Answer 35

You can make a table of pathogens and their different results from tests eg (indole test) so you form patterns with different pathogens

Question 36

``` 36. On a Antibiotic sensitivity plate testing, what do the following letters stand for? TE E P C VA CTX ```

Answer 36

``` TE = Tetracyclin E = Erythromycin P = Penicillin G C = Chloramphenicol VA = Vancomycin CTX = Cefotaxime ```

Question 37

37. How would you determine whats causing food poisoning in a patient?

Answer 37

1. Take a sample of stool 2. 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

Question 38

38. Why cant we culture viruses the same way we culture bacteria?

Answer 38

We cant grow viruses because they have intracellular | Viruses are defined that they NEED hosts cells

Question 39

39. How can we grow/culture viruses? | * we dont usually do this anymore**

Answer 39

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

Question 40

40. Aside from "Culture and Microscopy" how else can we see viruses?

Answer 40

Direct Antigen Detection | -ELISA test eg for influenza virus

Question 41

41. How can we use ELISA to detect influenza virus?

Answer 41

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

Question 42

42. Compare the following ways to detect infuenza: -Electron Microscopy -Culture -ELISA ?

Answer 42

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

Question 43

43. What are some advantages of Classical culture and identification?

Answer 43

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

Question 44

44. What are some disadvantages of Classical culture and identification?

Answer 44

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)