Systems of Pathogen Detection I

Question 1

Why is taxonomy better than standard naming for pathogens?

Answer 1

Names only imply capacity for pathogenicity

They do not include an assessment of virulence

Question 2

Outline the taxonomy of TB

Answer 2

Domain 	Eubacteria 
Kingdom Procaryote 
Phylum 	Gram positive 
Class 	Actinobacteria 
Order 	Actinomycetales 
Family 	Mycobacteriaceae 
Genus 	Mycobacterium 
Species 	Mycobacterium tuberculosis 
Strain 	Beijing 
Type 	IIa6B1 
Isolate 	Sputum

Question 3

Of the myobacterium genus which are pathogenic to humans?

Answer 3

148 current species - Only three are obligate human pathogens:

• M .tuberculosis
• M .leprae
• M .ulcerans

Question 4

Define a pathogen

Answer 4

A microbe capable of causing a specific degree of host damage

Question 5

What is a commensal non pathogen (in host)?

Answer 5

PRESENT but NOT CAPABLE of causing disease in the host
eg. E.coli Bacteroides thetaiotaomicron
‘good bacteria’

Question 6

What is a Zoonotic Non pathogen (in carrier)?

Answer 6

PRESENT but only CAPABLE of causing disease in ANOTHER host

eg. E.coli O157:H7 is subclinical in cattle

Question 7

What is a commensal opportunist (in host)?

Answer 7

PRESENT and CAPABLE of causing disease in the host but only in certain circumstances
eg. Bacteroides fragilis Coagulase Negative Staphylococcus (CNS)

Question 8

Why does a positive sample not confirm disease?

Answer 8

Being infected / presented with disease doesn’t guarantee active disease

Majority people presented with a pathogen don’t develop infection at all or it becomes latent and activated later on in life

Question 9

What determines how good a test result is?

Answer 9

A test result is only as good as the sample provided

Question 10

Describe the requirements of a sterile testing site

Answer 10

Sterile sites must be free from contamination

e.g. Skin flora in blood cultures

Question 11

How must non-sterile sites of testing be prepared?

Answer 11

Non sterile sites require decontamination of normal flora

e.g. Faeces, Mouth, Skin

Question 12

Why are some samples concentrated?

Answer 12

Samples with high volume or relatively low infected pathogen load require concentration (centrifugation, filtering)
e.g. CSF, Ascites, 24 hr Urine

Question 13

Why do we culture pathogens?

Answer 13

Culturing the bacteria proves it’s presence

But don’t always require culturing but knowing which specific pathogen you’re looking for makes identification a lot easier

Question 14

How is a culture prepared?

Answer 14

enrichment
purification
amplification

Question 15

How is a direct sample prepared for identification?

Answer 15

concentrated and treated

Question 16

What identification techniques are used to identify pathogens in a sample?

Answer 16

Molecular DNA/RNA
Microscopy: gross morphology
HPLC MassSpec: chemical Composition

Question 17

Which pathogens are visible via direct light microscopy?

Answer 17

Larger pathogens

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

Question 18

When is Electron microscopy used?

Answer 18

Smaller sized samples
Mainly used for viruses
Used when PCR and culturing isn’t an option

> not used for diagnostics

Question 19

Which pathogens are viewed using electron microscopy?

Answer 19

• Rotavirus (faeces)
• Rabies lyssavirus (brain tissue)
• Hepatitis B (liver)
• Tonsilitis adenovirus - nasal secretion

Question 20

When are samples stained?

Answer 20

Stain direct bacterial samples to identify using light microscopy

Question 21

What does staining show us?

Answer 21

Can identify:

• Gram stain
• Shape
• Features (flagella etc.)
• Gram +/-

Question 22

What is the significance of bacterial staining?

Answer 22

We can also use this staining to identify capsulated organisms as these are more likely to be pathogens

Question 23

Give examples of capsulated bacteria

Answer 23

• streptococcus pneumoniae

Bacterial spores also present in Bacillus anthracis and Clostridium perfringens (food poisoning)

Question 24

How is immunofluorescent staining done?

Answer 24

Immunofluorescent staining with pathogen specific conjugated antibody

Question 25

Which pathogens are seen using immunofluorescence?

Answer 25

Treponema pallidum in gastric syphilis Measles virus Infected into a lab Vero cell line then stained with fluorescent antibody

Question 26

What are the advantages of microscopy evaluation?

Answer 26

- Easy to perform - Rapid screening - Some parasites have SPECIFIC morphology eg. Schistosoma mansoni - Specific Immunofluorescence staining possible

Question 27

What are the disadvantages of microscopy evaluation?

Answer 27

- 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 28

What does a bacterial culture depend upon?

Answer 28

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

Question 29

What media are used for bacterial culturing?

Answer 29

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

Question 30

What are the different selective atmospheres?

Answer 30

- aerobic culture - anaerobic culture - microaerophilic culture

Question 31

Which pathogens favour aerobic atmospheres?

Answer 31

espiratory pathogens are adapted to this environment But these are both facultative anaerobes Strict (Obligate ) aerobe eg. Pseudomonas fluorescens, Nocardia asteroides)

Question 32

Which pathogens favour a microaerophilic atmosphere?

Answer 32

Respiratory pathogens - Neisseria meningitidis - Neisseria gonorrhoeae - Haemophilus influenzae - Brucella melitensis

Question 33

Why do certain pathogens require an anaerobic environment?

Answer 33

Some pathogens are unable to survive in aerobic conditions due to the effects of oxygen on their metabolism

Question 34

Which pathogens favour anaerobic conditions?

Answer 34

- Clostridium tetani - Clostridium botulinum - Clostridium difficile - Bacteroides fragilis

Question 35

Describe the effects of clostridium perfringens

Answer 35

Clostridium perfringens on Blood agar Grown in ANAEROBIC atmosphere Aerotolerant anaerobe producing spores in environmental conditions and exotoxins in humans causing Food poisoning (gut) and Gas gangrene

Question 36

Describe the structure of clostridium perfringens

Answer 36

Gram +ve spore forming anaerobic rod/bacillus shaped

Question 37

Which pathogen has selective atmosphere and temperature?

Answer 37

E.g. Campylobacter (found in cows) only grows in the following conditions: - 42°C - 10% CO₂

Question 38

What are the 2 types of specific haemolysis pathogens cause?

Answer 38

β- haemolysis | 𝛂-haemolysis

Question 39

Which pathogens cause βhaemolysis?

Answer 39

streptococcus sp. Group A

Question 40

Which pathogens undergo 𝛂-haemolysis ?

Answer 40

Mucoid: optochin sensitivity with gram stain microscopy and colony morphology - diagnostic for streptococcus pneumoniae

Question 41

Outline the different environments and pathogens of bacteriology

Answer 41

Environment - O₂ E. coli - CO₂ Haemophilus influenzae - ANO₂ Clostridium perfringens

Question 42

What info does bacteriology provide us?

Answer 42

Quantification, Identification, Antibiogram ``` Colony morphology, Colour, Haemolysis Colony Count Colony Identification Systematic identification Colony Resistance to antibiotics ```

Question 43

What is the purpose of metabolic testing?

Answer 43

Can identify Catalase enzymes Can cleave indole from tryptophan (indole test)

Question 44

How is bacteriology classified?

Answer 44

Specific patterns associated with certain pathogens - allows identification > taxonomically identified

Question 45

Give examples of some of the patterns used to classify pathogens

Answer 45

Metabolic function and sugar utilisation tests for identification of Enterobacteriaceae Eg. Salmonella, Shigella, E.coli Bacteria are particularly susceptible to phages (bacteriophages) ∴ some systems are used to phage type bacteria particularly E.coli

Question 46

When is antibiotic sensitivity plate testing carried out?

Answer 46

Only done if organism is able to be grown quickly

Question 47

What does an E test show?

Answer 47

Determines how much antibiotic to administer

Question 48

Which bacteria can cause food poisoning?

Answer 48

Can be caused by: - Shigella - Campylobacter - Salmonella

Question 49

What are the symptoms of food poisoning?

Answer 49

All these organisms produce similar illnesses : diarrhoea, vomiting, fever etc.

Question 50

How is the pathogen causing food poisoning identified?

Answer 50

Fecal samples are taken and tested in aforementioned ways to establish which pathogen is causing disease

Question 51

How is viral culture and microscopy conducted?

Answer 51

``` Requires permissive cell lines E.g. Vero cells (kidney epithelial) For Herpes Simplex Cytopathic effect Immunofluorescent staining of culture ```

Question 52

What are the methods of viral classical culture and identification?

Answer 52

Culture & Microscopy | Direct Antigen Detection

Question 53

What direct antigen test is used to identify viruses?

Answer 53

Rapid ELISA for fluA antigen = 15 mins ELISA for Flu Antibody

Question 54

How is viral cytopathic effect seen?

Answer 54

Through culture of permsisive cell lines Staining allows us to see cytopathic effects of virus - enables identification Not v. common anymore V. long and difficult to conduct

Question 55

Why is electron microscopy not enough to identify viruses?

Answer 55

Electron microscopy can see this virus but not identify it as ‘swine flu’ Culture takes 3-10 days

Question 56

Describe the outer protein structure of Influenza virus

Answer 56

The flu virus contains RNA on inside and 2 types of protein coat the outside (H and N)

Question 57

Outline how an ELISA for flu is conducted

Answer 57

1. Produce antibodies against H + N proteins 2. Place antibodies on a microtiter plate f 3. Capture virus and add to antibody + conjugate 4. Conjugate itself has enzyme attached that binds to substrate (virus) to fluoresce / colour

Question 58

What is a titre in serological ELISA?

Answer 58

dilution of sample that gives a signal equal or greater than the positive control cut off

Question 59

What are the advantages of classical culture and identification?

Answer 59

- Cheap simple, reliable reagents - Sensitive; single organisms grown and identified - Validated specificity; ‘Gold Standards’ w/ multiple parameters - Direct in vivo measurement of effectiveness of therapy eg Antibiotic sensitivity - Easily archived; epidemiology

Question 60

Outline disadvantages of classical culture and identification

Answer 60

- Some pathogens cannot be grown eg. Mycobacterium leprae - Some pathogens aren't 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. Mycobacterium tuberculosis - Labour intensive (expensive) - Requires specialist interpretive expertise (more expensive)