10- System for Detecting Pathogens I

Question 1

what is a pathogen?

Answer 1

microbe capable of causing a specific degree of host damage

Question 2

what is a commensal non-pathogen

Answer 2

present in host - not capable of causing disease in the host

part of everyday life - e.g. E. coli in gut

Question 3

what is a zoonotic non-pathogen

Answer 3

present and commensal in an animal, only capable of causing disease in another host

Question 4

what is a commensal opportunist

Answer 4

present and capable of causing disease in the host but only in certain circumstances - e.g. with increased susceptibility to disease, immunosuppression

Question 5

why is it that not all samples that are positive are actually diagnostic of active disease

Answer 5

not all pathogens cause disease

may reflect past infections, subclinical infections, or non-pathogenic colonization, rather than active disease

Question 6

what are the complications of taking a sample to test for a pathogen?

Answer 6

• sterile sites must be free from contamination = e.g. ethanol swabs to reduce/ kill skin flora before a blood test, avoids contamination
• non-sterile sites require decontamination of normal flora = e.g. background flora in faeces needs to be decontaminated
• samples with high volume or relatively low infected pathogen load require concentration via centrifugation or filtering
  = infections at a low viral load, harder to detect and needs to be concentrated

Question 7

list preparation techniques before culturing a micro-organism

Answer 7

enrichment
purification
concentration of sample
treating sample to get rid of unwanted background micro-organisms

Question 8

list different techniques following culturing for identifying the organism

Answer 8

extracting molecular DNA/ RNA for analysis and identification

looking at gross morphology using light/ electron microscope

chemical composition analysed through various biochemical tests

Question 9

list different techniques that allow for the detection of bacterial pathogens using microscopy

Answer 9

direct light microscopy for big organisms - easily identifiable

direct electron microscopy for smaller organisms

direct bacterial staining of samples for bacteria - staining to identify gram neg/ positive (red/ pink for g-neg; purple for g-pos)

liquid stain to identify encapsulated bacteria - these are good at evading the immune system

immunofluorescent staining with pathogen specific conjugated antibody - for pathogens that don’t gram stain well

Question 10

advantages of microscopic methods for bacteriology

Answer 10

easy to perform

rapid screening

some parasites have specific morphology – e.g. Schistosoma mansion

specific immunofluorescence staining is possible

Question 11

disadvantages of microscopic methods for bacteriology

Answer 11

not sensitive

hard to pick up organisms that cause disease at a low infectious dose - e.g. screening sputum smears requires at least 10,000 organisms per ml

general stains aren’t specific

labour intensive and expensive

requires specialist interpretive expertise (more expensive)

Question 12

what conditions must be considered for classical bacterial culture and identification

Answer 12

media/ type of agar- non-selective, semi-selective or selective = CLED, TSC…

atmosphere - no oxygen/ anaerobes, high CO2 (resp pathogens), aerobes/ high O2, microaerophilic

temperature

Question 13

example of non-selective (agar) media?

Answer 13

blood agar

Question 14

example of semi-selective media? what grows on these?

Answer 14

MacConkey - for gram neg enteric bacteria

CLED - e.g. for non-lactose fermenting Enterobacteria (Shigella and Salmonella from faecal sample)

Question 15

what bacteria grow in aerobic culture?

Answer 15

grow with oxygen

e.g. S. aureus, strict obligate aerobes

Question 16

what is microaerophilic culture? examples of organisms that grow in it?

Answer 16

microaerophilic = low levels of dioxygen present in environment for optimal growth

resp pathogens like Neisseria meningitidis & Neisseria gonorrhoea
- lung pathogens grow better in high CO2/ low O2 concs = environment of lung is similar

Question 17

what is anaerobic culture? examples of organisms that grow in it?

Answer 17

grow in absence of O2

e.g. Clostridium difficile

Question 18

describe the conditions for Campylobacter growth - how does it affect humans?

Answer 18

42 degrees, 10% CO2
- chicken temperature

in humans - releases toxin, prevents water being absorbed from colon to blood = water diarrhoea

Question 19

what are the two types of haemolysis of blood

Answer 19

beta haemolysis
alpha haemolysis

Question 20

what is beta haemolysis?

Answer 20

complete lysis of RBCs = creates clear, colourless zone surrounding colonies

e.g. Streptococcus pyogenes (group A)

often pathogenic streptococci cause beta haemolysis – causes strep throat

organisms are after the iron in haem of RBCs to grow

Question 21

what is alpha haemolysis?

Answer 21

partial/ incomplete lysis of RBCs = causes greenish-brownish discoloration of agar

e.g. streptococcus pneumonia/ viridians

bacteria that exhibit alpha haemolysis are often part of the normal mouth and throat flora, can cause opportunistic infections too

Question 22

list different techniques/ conditions that help with classical culture and identification

Answer 22

growth in different environments - media, oxygen/CO2/ANO2 conditions, temperature

type of haemolysis

colony count, identification

chemical tests - enzymes produced

antibiotic sensitivity tests - resistance to antibiotics

sugar tests - monitor growth in different sugars

Question 23

what colour is alpha haemolysis?

Answer 23

greenish/brown

Question 24

what colour is beta haemolysis?

Answer 24

clear/ colourless - opaque white

Question 25

what does MacConkey agar grow?

Answer 25

gram negative bacteria, specifically enteric bacteria

Question 26

what do CLED plates grow?

Answer 26

micro-organisms in urine samples

Question 27

what does blood agar grow?

Answer 27

to grow bacteria and differentiate them based on haemolytic properties

e.g. Haemophilus influenzae, Streptococcus pneumoniae and Neisseria species = hard to grow on normal agar

Question 28

what does chocolate agar grow?

Answer 28

differential medium for gram-positive cocci - gram positive or negative

Question 29

understand the concept of systematic bacteriology

Answer 29

classification, identification and taxonomy of bacteria

classifying them into groups based on shared characteristics - helps understand their pathogenic potential

builds a profile based on various tests for the pathogen - specifically down to species and strain

Question 30

what are the two types of antibiotic sensitivity plate testing?

Answer 30

zones of inhibition by various antibiotics on one agar plate - determines antibiotic resistant strains and the antibiotic they’re resistant to

E-test = determines the ideal dosage/ conc and course of an antibiotic based on at what conc the antibiotic starts visibly killing the cultured bacteria

Question 31

what are the methods involved in classical culture and identification for virology?

Answer 31

permissive cell lines after culture to observe cytopathic effects

immunofluorescent staining of culture

direct antigen detection via ELISA

Question 32

describe visualising cytopathic effects in permissive cell lines

Answer 32

growing cells in permissive cell lines that support that specific virus’ growth

cytopathic effects are observable chances/ damage in host cells due to viral infection - e.g. cell lysis, rounding, detachment, formation of syncytia/ giant multinucleated

Question 33

describe immunofluorescent staining of a viral culture

Answer 33

infected cell cultures fixed and stained with specific antibodies – bind to viral proteins

antibodies are tagged with fluorescent dyes detect viral antigens in infected cells - observe fluorescence under microscope

Question 34

describe direct and serological ELISA

Answer 34

detects specific viral antigens in patient sample- virus needs to be known/ suspected beforehand

specific antibody against the viral antigen is coated on a plate – capture antibody

patient sample is added = if the antigen is present it binds to the capture antibody

second antibody – detection antibody – linked to an enzyme binds to the antigen

substrate for enzyme is added – causes a colour change that can be measured

Question 35

advantages of virology & bacteriology culture and identification techniques

Answer 35

cheap, simple, reliable reagents

sensitive - single organisms can be grown and identified

validated specificity - e.g. ELISA, specific antibody-antigen binding

direct in vivo measurement of effectiveness of therapy - e.g. antibiotic sensitivity testing

Question 36

disadvantages of virology & bacteriology culture and identification techniques

Answer 36

some pathogens can’t be grown

some pathogens cannot be well differentiated by biochemistry alone

slow: culture requires at least overnight incubation
- viral = 3-10 days, mycobacterial = 6-12 weeks

labour intensive, expensive, requires specialised interpretation