System For Detection Of Pathogens I

Question 1

What is a pathogen?
State the 3 types of pathogens present?

Answer 1

• Commensal Non pathogen (in host): PRESENT but NOT CAPABLE of causing disease in the host eg. E.coli Bacteroides thetaiotaomicron ‘good bacteria’
• Zoonotic Non pathogen (in carrier): PRESENT but only CAPABLE of causing disease in ANOTHER host eg.E.coli 0157:H7 is subclinical in cattle
• Commensal Opportunist (in host): PRESENT and CAPABLE of causing disease in the host but only in certain circumstances eg. Bacteroides fragilis, Coagulase Negative Staphylococcus (CNS)

Question 2

Are all positive samples of a pathogen, a diagnostic of disease?

Answer 2

• No either in state of:
• No infection
• Infection: Latent (may cause disease), but currently no symptoms, Active infection

Question 3

State the definition of a pathogen?

Answer 3

A microbe CAPABLE of causing a specific degree of host damage

Question 4

What measures should you take in terms of the sample provided to ensure max. efficacy of test result?

Answer 4

• Sterile sites must be free from contamination eg. Skin flora in blood cultures.
• Non sterile sites require decontamination of normal flora eg Faces, Mouth, Skin.
• Samples with high volume or relatively low infected pathogen load require concentration (centrifugation, filtering) eg CSF, Ascites, 24 hr Urine.

Question 5

Describe the steps of the prep phase and identification phase of direct and culture?

Answer 5

VD

Question 6

What is light microscopy typically used for?

Answer 6

• Large microbes: examples include
• Trichomonas vaginalis: 160 Million people infected
• Schistosoma mansoni: 83 Million people infected
• Entamoeba histolytica: 50 Million people infected
• Strongyloides (threadworm): 50% UK children

Question 7

What is electron microscopy used for?

Answer 7

• Small microbes like viruses: examples include:
• Rotavirus (Reovirus) from Faces
• Rabies (Lyssavirus) from Brain tissue
• Hepatitis B (Hepadnavirus) from Liver
• Tonsilitis (Adenovirus) from Nasal sec

Question 8

What is staining of samples used for?

Answer 8

• To view Bacterial Features like capsule or bacterial spores

Question 9

State advantages of microscopy?

Answer 9

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

Question 10

State disadvantages of microscopy?

Answer 10

• 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 11

Why does the culture rely on bacteria in order to work?

Answer 11

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

Question 12

State the 3 types of media found within cultures with examples?

Answer 12

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

Question 13

Describe selective media?

Answer 13

Selective media will only grow the pathogens using a potentially mixed sample (e.g. faecal or urines).

Question 14

Describe the use of selective atmopshere and the different cultures that can be used? (PART 1)

Answer 14

• A selective atmosphere of growth can differentiate pathogens from one another (e.g. aerobic and anaerobic organisms) -> So are obligate to their environment of growth whilst others are not.
• 1. Aerobic culture: S. aeureus, Catalase positive, E.coli
• 2. Microaerophilic culture - may use oxygen when carrying out aerobic respiration. They live in an environment where oxygen level is low.
• 3. Anaerobic culture: eg. Clostridium tetani, Clostridium botulinum, Clostridium difficile, Bacteroides fragilis

Question 15

Describe the use of selective atmopshere and the different cultures that can be used? (PART 2)

Answer 15

• Some organisms grow in a microaerophilic system whilst others are respiratory pathogens.
• Capnophilic pathogens require carbon dioxide for growth whilst obligate anaerobes are killed by oxygen as they do not have the capacity to deal with the effects of oxygen on their metabolism
• (these pathogens live in places like the gut and degrading tissue due to lack of oxygen supply in something like frostbite that leads to gangrene).
• TSC agar is used to grow obligate anaerobes. Some pathogens grow in selective temperatures with Campylobacter growing in 420 С.

Question 16

What bacteria can be used in specific haemolysis in blood?

Answer 16

• The haemolysis certain pathogens induce in blood can also be used to describe them with some inducing alpha haemolysis
• Where RBCs are partially broken down leaving behind a green colour and others inducing beta haemolysis
• where RBCs and haemoglobin is broken down completely leaving a clear zone around bacterial growth.

Question 17

What type of environment (gas wise) does E.coli, haemophilus
influenzae and clostridium perfingens grow in?

Answer 17

• O2: E. coli
• CO2: Haemophilus influenza
• ANO2: Clostridium perfringens

Question 18

Describe the factors used in the quantification, identification and antibiogram in classical culture and identifcation?

Answer 18

• Colony morphology, Colour, Haemolysis
• Colony Count
• Colon Identification
• Systematic identification
• Colony Resistance to antibiotics

Question 19

State what type of test and the name of tests can be used for the presence of E.coli and clostridium perfingens?

Answer 19

• Classical metabolic testing
• Catalase: E.coli = +ve, Clostridium perfringens = -ve
• Indole test: Can cleave indole from tryptophan
• E.coli = +ve, Clostridium perfringens = -ve

Question 20

What is systemic bacteriology and what type of bacteria can it be used to identify?

Answer 20

• These different environmental, antibiotic sensitivity, metabolic and morphology tests can all be collated into a system to identify the organism via a taxonomy table (table with example shown right).
• This process known as systematic bacteriology is a well tried process of identifying pathogens.
• Metabolic function and sugar utilisation tests for identification of Enterobacteriacae Eg. Salmonella, Shigella, E.coli

Question 21

What is the use of phage typeing test?

Answer 21

• Phage typing tests the susceptability of an organism to the bacteriophage
• Phage = virus that only affects the bacteria

Question 22

What is the Use of antibiotic sensitivity testing?

Answer 22

Used to test for which drugs to adminster

Question 23

What does the culture in virology require?

Answer 23

• Requires permissive cell lines eq.Vero cells (Kidney epithelial) for Herpes simplex
• Cytopathic Effect
• Immunofluorescent staining of culture

Question 24

What can be used to identify viruses?

Answer 24

• Culture & microscopy
• Direct Antigen Detection via ELISA eg. Influenza Virus

Question 25

Describe the use of ELISA in influenza virus?

Answer 25

• ELISA tied to specific antigens can be measured by
• Multiple samples single antigen with signal positive colour cut off
• Multiple antigens but serial dilution of sample

Question 26

State advantages of using classical systems?

Answer 26

• Cheap simple, reliable reagents
• Sensitive: 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 27

State disadvantages of using classical systems?

Answer 27

• Some pathogens cannot be grown eg. 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: eg. Mycobacterium tuberculosis
• Labour intensive (expensive)
• Requires specialist interpretive expertise (more expensive)