Molecular and Genomic Epidemiology of Infections

Question 1

1. What is molecular Epidemiology?

Answer 1

A resolved measure (diversity) of differences (variables)
that determines:
a) Disease distribution in time and place
b) Disease transmission
c) Disease manifestation
d) Disease progression

Question 2

2. What kinds of questions can molecular epidemiology answer?

Answer 2

• -> Confirming Outbreaks ( eg source of pathogen, geographical spread, is it actually an outbreak or is it a contamination)
• > Identifying Disease risks (virulence changed? resistant strains ? new infection or recrudescence of old one?)

Question 3

3. How much difference is enough to tell if a pathogen is present?

Answer 3

We need to look at molecular changes , and see why is there changes (resolving diversity).

Question 4

4. What are the three functional characteristics to determine what pathogen you’re looking for?

Answer 4

• Classical = Biochemistry
• Serology = how is the host affected
• Virulence = Verotoxins

Question 5

5. What genomic characteristics might we look at?

Answer 5

look at DNA – how many genes do we look at?
—Resistant genes
Look for changes that caused by genes – phenotypes. Or look at amino acids->protein -> see how protein is affected . look at base sequence eg SNPs – maybe make it more virulent or less virulent. Look at Ribosome or miRNA.,
-Changes in translocations/re-arrangements – happens during transmissions –
Looks of targets to look at

Question 6

6. What is the resistant gene in rifampicin , ans what does this treat?

Answer 6

rpo gene is the resistant gene in rifampicin that we treat with MDR TB.

Question 7

7. What is the difference between single weighting and additive weighting?

Answer 7

single weighting = Just looking at whether the pathogen is there or not through one test eg for test for antigen or verotoxin

Additive = Do many tests - know exactly what the organism is and how virulent it is

Question 8

8. What do you know about the  0157 e.coli? 
ie symptoms
toxin produced
how we test for it
what type of weighting?

Answer 8

0157- Diarrhoea Vomiting. Produces toxin – O157 Toxin ( 2+ve) – grow it on media – grows colonies- can sub culture colonies – do a latex test with antibodies on blue latex beads –> show gene (stx2) is there using PCR ,do phage testing – to give us a phage number 21/288
additive weighting of single tests (multiple single tests)

Question 9

9. How did scientists investigate an epidemic outbreak of E.Coli in Godstone farm 2009?

Answer 9

-kids were getting ill
-we need to know if cases are the same organism , which organism, same phage type, same toxin
-To close down zoo- hazard
-Which animals are infected
Tests are done ,
Tells us where its from- sheep(poo-straw-positive samples found using VTEC)
Wasn’t in sand pit or water – we know it wasn’t coming through water

Question 10

10. What do you look at when you do multiple weighting for factoral genomic factors?

Answer 10

Presence or absence of a gene/base/s change

in genome/gene relative to location in the genome

Question 11

11. How we can test using multiple weighting for TB?

Answer 11

We can use Spoligotyping  (rapid PCR , tells us strains of TB)
 1. PCR with
RE region primers
generates multiple length
amplicons

2.Hybridization of labelled PCR products onto
43 spacer specific oligonucleotides (between RE sequences)
fixed on a membrane then visualise signal with RE probe

Result is a profile of the presence/absence of specific repeats at ONE locus

Question 12

12. What can we do with the results from a spoligotyping?

Answer 12

Make a dendrogram that shows relatedness of patterns

Question 13

13. One other system for detecting factoral multiple copy number systems is VNTR, what is this?

Answer 13

Variable Number of Tandem Repeats (VNTR)

The result of this is a profile of the number of specific repeats at multiple genomic loci

Question 14

14. How can you use multiple weighting of functional genomic factors to detect pathogens?

Answer 14

You can look at the types of substitutions that occur in the genome (eg synonymous or not)
ie important to note that a small amount of changes to bases can result in lots of different types of aa->proteins->organism

Question 15

15. What are silent mutations?

Answer 15

Mutations that are Intragenic (between genes)
or Synonymous (not altering coding)

Question 16

16. What are non-synonymous mutations?

Answer 16

Substitutions causing coding to be altered

Question 17

17. What are some examples of corruptive mutations?

Answer 17

Deletions or Insertions (disrupting coding frame)
Creation of STOP codons (truncation)
Corruption of STOP codons (elongation)
Corruption of CONTROL sequences (eg. promoters)

Question 18

18. What is the sequence AAA?

Answer 18

STOP codon

Question 19

19. What is DRIFT?

Answer 19

• > Gradual alteration in sequence

- > These drifts mean that maybe a mutation could not allow present antibodies made to bind to their pathogens antigen

Question 20

20. Why cant vaccines completely wipe out a pathogen?

Answer 20

Herd immunity (after large vaccination program)
kills most but also selects for escape mutants that
maintain the drift --> people who were once immune wont be immune anymore

Question 21

21. What is antigenic drift?

Answer 21

Its when an antigen keeps getting its sequenced changed by drift and eventually wont bind to antibodies
*SAME ANTIGEN–> CHANGED. NOT NEW ANTIGEN**

Question 22

22. What does multiple weighting of temporal genomic factors tell us?

Answer 22

Tells us the mutation rate ( the time since the last alteration)

• Bases are changing but maybe a different time between changes
• You would imagine that change likelihood is constant

Question 23

23. What is this idea of a “ Constant Molecular Clock” ??

Answer 23

Accurate predictions in molecular epidemiology thus requires
an assumption that evolution is driven by a
constant molecular clock

Question 24

24.How does mutation contribute to evolution?

Answer 24

Diversity progresses because random mutations occurring at a regular rate

Question 25

25. What are 5 factors affecting the speed of the 'molecular clock'?

Answer 25

1. Replication Rate 2. Proof Reading Enzyme 3. Pressure from the host 4. Redundancy 5. Transmission Rate

Question 26

26. How does replication rate affect the molecular clock?

Answer 26

A high division rate provides a higher mutation rate

Question 27

27. How does polymerase | proof reading fidelity affect the molecular clock?

Answer 27

Some species (eg HIV) have low fidelity promoting high mutation rate

Question 28

28. How does Selection pressure from the host or environment affect the molecular clock?

Answer 28

High selection pressure removes ‘weak’ mutants and emphasises clusters Loss of selection pressure allows deletions

Question 29

29. How does the Degree of redundancy in the genome affect the molecular clock?

Answer 29

multiple copies of a single gene in the genome allow for mutations in one copy without compromising overall functionality Movement or recombination within genome may not effect phenotype

Question 30

30. How does transmission rate affect the molecular clock?

Answer 30

High transmission rates relative to the mutation rate | results in dissemination and single strain outbreaks

Question 31

31. Which virus has a high transmission rate and why?

Answer 31

Influenza virus | eg Flu A , because there are 2-3 bases changing per year there is a 1.5 transmission per infection

Question 32

32. What caused the bird flu outbreak in 1990s?

Answer 32

-> Antigenic Drift of Haemagglutinin and Neuraminidase antigens

Question 33

33. Which types of genes are most likely to change?

Answer 33

Hyper-variable genes change more rapidly than conserved genes but Conserved genes are more likely to be associated with phenotype and virulence

Question 34

34. Do all changes result in a new organism?

Answer 34

Not all changes are new | Some may revert BACK to an older profile (convergent evolution)

Question 35

35. Large and rapid changes are rare in genes but why would this be particularly useful to a pathogen?

Answer 35

Large and rapid changes are rare | but often lead to escape from existing herd protection

Question 36

36. What is antigenic shift?

Answer 36

Antigenic shift is a sudden replacement of an antigen by recombination with another viral type that has evolved separately (either in another animal or another human population). New types will not be protected against by previous infection or vaccination - leading to new epidemics.

Question 37

37. Difference between antigenic drift and antigen shift?

Answer 37

antigenic drift - same antigen , some genomic changes, immunity might still work Antigenic shift - new antigen with combination of another viral protein. No immunity

Question 38

38. How can we test transmission effectively for eg in hospital acquired infections

Answer 38

- Look at molecular restriction digest typing = monitor effectiveness of control measures (DNA extracted and cut with the same restriction enzymes then pieces separated on a gel then visualised)

Question 39

39. How can we see where reservoirs of infection are? | * 2 ways*

Answer 39

Through contact tracing eg one person with HIV spreading it to another two- who did they spread it too Determining the introduction events - pinpointing the first case and where it all began from eg ebola first case in Guinea

Question 40

40. What can we look foe when we're trying to find the spread or emergence of resistance?

Answer 40

-Drug resistance polymorphisms | -

Question 41

41.Molecular epidemiology offers a variety of methods to test questions Involving disease transmission, strain virulence, pathogen evolution Choosing the most appropriate system requires...?

Answer 41

1. Knowing the most appropriate variable/s 2. Quantitating variations and deriving diversity 3. Generating identities or clusters 4. Applying related data geographic location time of isolation incidence prevalence transmission rate severity of disease