Molecular and Genomic Epidemiology of Pathogens

Question 1

What is molecular epidemiology?

Answer 1

• A resolved measure (diversity) of differences (variables).
• We resolve the measure of diversity by doing a tests at any time in any disease. These will answer certain things depending on the asked questions.

Question 2

Example of questions asked in molecular epidemiology

Answer 2

1. How disease is distributed in time and place?
2. How is the disease transmitted?
3. How does the disease manifest?
4. What is the disease progression? acute or chronic? change in host and phenotype?
   There are many things we look at depending on the questions.

Question 3

What questions are asked to confirming outbreaks?

Answer 3

• In institutions: which patient caught the pathogen? etc
• In the community: Who was the index case and what is the likely source?
• In the past: What has driven the geographical spread of important strains? Identify how significant a strain is.
• In the lab: Is this an outbreak or a contaminant?

Question 4

How is the shift in virulence looked at?

Answer 4

• Has the incidence of annual infections increased from last year?
• Common antibiotics means that some infections will be resistant in the future making a person untreatable
• For example, the flu virus can be used to predict which strains are coming to make a vaccine in advance.

Question 5

How are questions about whether an infection is new or recrudescence be answered?

Answer 5

By looking at molecular changes/constants that happen within pathogens.

• Which variable? (TARGET)
• How many variables? (TARGETS)
• How much of a difference is enough? (RESOLVED DIVERSITY)

Question 6

What are the functional ways that we can resolve diversity?

Answer 6

Classical
Serology
Virulence

Question 7

What is another way that we can resolve diversity?

Answer 7

By looking genomic characteristics

Question 8

What else can epidemiology be used to look at?

Answer 8

Can also look at the rearrangement of pathogen genes which is important to look at.

Question 9

Single weighting

Answer 9

The presence or absence of a toxin can be identified through a biochemical test, presence of O157 antigen or a verotoxin.

Question 10

Additive weighting

Answer 10

Combination of single tests to make sure we know exactly what type of organism, how virulent the organism, whether the organism has changed etc.

Question 11

Multiple weighting

Answer 11

Genomic factors that affect the level of diversity e.g. factorial (presence or absence of a gene based on gene relative to location), functional (type of substitution) and temporal (mutation rate).

Question 12

E. coli O157 Additive weighting of Single Testing

Answer 12

1) First single test - culture on selective media: can be grown into colonies and be subcultured.
2) Second single test - Latex test: Take the colonies and do O157 serotyping using antibody on blue latex beads. Identifies which colonies are producing the colonies.
3) Third Single Test - PCR of verotoxin gene (stx 2): In E. coli, we can do gene testing to show that the stx 2 gene is missing in some strands.
4) Fourth Single Test: Phage typing (Pt 21/28): Can be done to show what phage types the E. coli are.

All these tests together allow us to name the pathogen and to gather more information on the pathogen.
Identification and typing of E. coli O157: verotoxin 2 +ve: Phage type 21/28

Question 13

Godstone farm petting zoo example

Answer 13

• In 2009, children who had visited the farm were getting sick. This was increasing over time.
• It was important to know what pathogen was causing this and whether it was the same phage to make sure the strain was not changing. This was able to prove which animals were a hazard to human health.
• This was caused by E. coli and the secondary cases were caused by passing on from original sick children.
• It wasn’t present in water because it was caused by one of the sheep who had the E. coli and was pooping in the straw which was then spread around the farm.
• Tracing it back to the source allowed the infection to be contained. This is an example of why additive weighting or molecular epidemiology is used to identify the pathogen.

Question 14

What is multiple weighting?

Answer 14

Other factors can be combined and not just tests but also genomic factors.

Question 15

Factorial

Answer 15

Presence or absence of a gene/base/s change in genome/gene relative to location in the genome
- Looking at more than one gene can allow us to decide if the presene or absence of a gene is significant in disease causing.

Question 16

Spoligotyping

Answer 16

1) PCR with RE region primers generates multiple length amplicons
2) Hybridisation of labelled PCR products onto 43 spacer specific oligonucleotides (between RE sequences) fixed on a membrane than visualise signal with RE probe.

Question 17

TB and Spoligotyping

Answer 17

• This technique is used to identify the regions of the gene that are different for example presence or absence e.g. in tuberculosis - there is a region of the gene that has 43 copies of the same sequence.
• As TB spreads, the change of the genomic number will determine the strain of TB that the patient has.
• The result is a generation of a profile of the absence/presence of specific repeats at the ONE locus.

Question 18

Uses of Spoligotyping

Answer 18

• Look at relatedness of a pattern by looking at past correlations
• Look at prevalent strains in the world
• This pattern can be used to make a tree (dendrogram) to be able to see the taxonomic changes and relatedness between the strains of a disease.
• The distance between the strains indicates relatedness.

Question 19

What are other methods of factorial testing?

Answer 19

Variable number of tandem repeats (VNTR): to identify the changes in the genome of strains.
This involves the use of PCR. This is quicker than spoligotyping.

Question 20

Variable number of tandem repeats

Answer 20

1. Add a PCR primers at the different copies.
2. Identify the number of copies in a strain by looking at the PCR product.
3. If the no. changes, then identify the patterns and profiles to identify the type of strain.
4. This can then be used to make a dendrogram showing the relatedness of pattern.

Question 21

Functional Multiple Weighting

Answer 21

This involves looking at the type of substitution.

Question 22

6 outcomes and relatedness

Answer 22

• Lots of substitutions can occur - 3 mutations have occurred however, there are 6 outcomes.
• This schematic also shows the type of mutations that are closer in relatedness than others.

Question 23

What is the redundancy factor?

Answer 23

• This is complicated with redundancy in the way that mutations produce amino acids. The combination of the triplet code is more than one. This means that sometimes when there is a change in the base, it doesn’t necessarily change the amino acid.

Question 24

Why is the redundancy factor important?

Answer 24

This is important from a virulence or functional point of view.

Question 25

Silent change

Answer 25

• A synonymous change can be made but these mutations will not alter the coding.
• Mutations that are intragenic (between genes).

Question 26

Non-synonymous change

Answer 26

• Substitutions causing coding to be altered

Question 27

Corruptive change

Answer 27

• Deletions or insertions (disrupting the coding frame)
• Creation of STOP codons (truncation)
• Corruption of STOP codons (elongation)
• Corruption of CONTROL sequences (e.g. promoters)

Question 28

Example of functional diversity

Answer 28

The substitution is changed and it changes the amino acid. There are 4 possible changes. GATTTGA.

• 3 base changes can derive 4 possible 8AA types.
  - 2 with STOP terminators
  
  • 1 synonymous
  • 1 non-synonymous

Question 29

What is drifting? In terms of the flu virus?

Answer 29

Gradual alteration in sequence. In terms of the flu virus, change in sequence of the virus is drifting

Question 30

What can affect the way surface antigens are presented and Ab binding affinity?

Answer 30

• Some mutations will have more influence on the way amino acids are made than others. This affects the certain types of ways that the surface antigens are presented.
• Some mutations have more influence on Ab binding affinity than others.

Question 31

When does the flu virus mutate?

Answer 31

• The flu mutation mostly occurs randomly - it will occur in the Haemaagglutin and Neuraminase gene. However, certain genes will be lost. This is because each change happens multiple times a year.

Question 32

What is herd immunity?

Answer 32

This is when over time, after large vaccination programs, such as the flu vaccine, this will kill most but also selects for the escape mutants that maintain the drift.

Question 33

What does herd immunity allow for?

Answer 33

• It allows for the identification of the mutation that will be the new strain of flu.. As it will be the only type that has ‘escaped’. ;

Question 34

Temporal Multiple Weighting

Answer 34

• Mutation rate (time since the last alteration)

- The molecular clock or the change likelihood is constant.

Question 35

What assumptions are made when a dendrogram is made?

Answer 35

• Made with the assumption that there is a ‘constant molecular clock’.

Question 36

What do accurate predictions in molecular epidemiology require?

Answer 36

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

Question 37

Why is it assumed that the molecular clock is constant?

Answer 37

• So that predictions can be made about disease spread and how quickly it navigates to different areas.
• It suggests that diversity progresses because the random mutations occur at a regular rate.

Question 38

What factors affect the speed of the “molecular clock”?

Answer 38

1. Bacterial replication rate: high division rate provides a higher mutation rate
2. DNA or RNA polymerase proofreading fidelity: low fidelity promoting high mutation rate
3. Selection pressure from the host or environment: Loss of selection pressure, allows deletions
4. Degree of redundancy in the genome: multiple copies of a single gene allows for mutations in one copy without compromising overall functionality.
5. Transmission rate: high transmission rates results in dissemination and single strain outbreaks

Question 39

Which genes change the most?

Answer 39

1. Hyper-variable genes more than conserved genes.
   1a) Conserved genes needed for survival, production and design. For phenotype and virulence.
2. Convergent evolution meaning changed genes revert back to older profile
3. Large and rapid changes are rapid but lead to escape from existing herd protection.

Question 40

What can result from antigenic shift?

Answer 40

Some genes could be replaced entirely.

Question 41

How does recombination occur in the flu virus?

Answer 41

Can occur when infected with multiple strains at a time, leading to a mixture between the two.

Question 42

What is antigenic shift?

Answer 42

A sudden replacement of an antigen by recombination with another viral type that has evolved separately (either in another animal or another human population).

Question 43

How does new epidemics occur due to antigenic shift?

Answer 43

New types will not be protected against by previous infection or vaccination - leading to new epidemics.

Question 44

How is molecular epidemiology used?

Answer 44

• Used to control the source of infection
• Resevoirs of infection: contact tracing -> find out where the source of infection is.
• Reservoirs of infection: determining introduction events -> find out where or how many outbreaks were occuring.
• Spread or emergence of resistance -> Look at whether resistance has occured or not.

Question 45

How can resistance be predicted?

Answer 45

By using certain antibiotics by looking at particular SNPs.