Molecular and genomic epidemiology of infections

Question 1

What is molecular epidemology and what does it determine?

Answer 1

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

Question 2

What kind of Q’s can ME answer?

Answer 2

• Confirming outbreaks for example:
• Inside institutions: Did patient A catch this pathogen from patient B? Do patients A, B & C from the same hospital ward have the same strain?
• 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?
• In the lab: Is this an outbreak or a contaminant?

Question 3

What kind of Q’s can ME answer?

Answer 3

• Identifying disease risks for example:
• Shifts in virulence: Has the incidence of annual infections increased from …last year? Are drug resistant strains on the rise? From where?
• Reservoirs of infection: New infection or recrudescence? (Breakout)

Question 4

Describe the factors taken into consideration when deciding what the target is and how many of them are in ME?

Answer 4

VD

Question 5

Describe the factors when deciding how much diversity is within ME of a disease?

Answer 5

1. Single Weighting (single test)
   a. Presence or absence (of a particular gene or toxin)
   i. Biochemical test
   il. Presence of 0157 antigen
   ii. Presence of Verotoxin
2. Additive Weighting: Combination of single tests
3. Multiple Weighting: Genomic factors

Question 6

State the tests involved in additive weighting of E.coli?

Answer 6

• Culture on selective media
• 0157 serotyping using antibody on blue latex beads
• PCR of DNA for verotoxin gene (stx2)
• Phage typing (PT 21/28)

Question 7

Multiple weighting diversity
State the 3 types of multiple weighting involved?

Answer 7

• Factoral: Presence or absence of a gene/base/s change in genome/gene relative to location in the genome
• Functional: Type of substitution (synonymous/non synonymous)
• Temporal: Mutation rate (time since the last alteration)

Question 8

State the 2 factoral multiple copy number systems that can be used for factoral multiple weighting diversity?

Answer 8

1. Spoligotyping - for one locus
2. Variable number of tandemic repeats (VNTR) -> for multiple loci

Question 9

Describe the process of spoligotyping?

Answer 9

• Step 1: PCR with RE region primers generates multiple length amplicons
• Step 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
• Spoligotyping dendogram showing relatedness of pattern

Question 10

What are Variable tandem repeats?

Answer 10

Variable number of tandem repeats (VNTR) is a location in DNA where a short nucleotide sequence is organized as a tandem repeat with variations in length between individuals.

Question 11

Describe variable number of tandemic repeats method?

Answer 11

• Perform PCR on the region, where number of copies on VTR determines the length of amplicon
• Result is a profile of the number of specific repeats at multiple genomic loci -> VNTR denedorgam shows relatedness of pattern

Question 12

Functional
State the 3 major types of single base substitutions in terms of their effects and how it affects function?

Answer 12

• Silent: Mutations that are Intragenic (between genes) or Synonymous (not altering coding)
• Non synonymous: Substitutions causing coding to be altered
• Corruptive: Deletions or Insertions (disrupting coding frame). Creation of STOP codons (truncation). Corruption of STOP codons (elongation). Corruption of CONTROL sequences (eg. promoters)

Question 13

What is genetic drift and how does herd immunity come into play?

Answer 13

• Gradual alteration in sequence
• Genetic drift is the change in the frequency of an existing gene variant (allele) in a population due to random chance
• Causes mutations to arise where some have more influence on AB binding affinity than others
• Herd immunity kills mosts strains, but selects for escape mutant that maintain drift.

Question 14

Temporal
What is the constant molecular clock?

Answer 14

Accurate predictions in molecular epidemiology thus requires an assumption that evolution is driven by a ‘Constant Molecular Clock’

Question 15

How is it involved in molecular epidemology?

Answer 15

• The molecular clock hypothesis states that DNA and protein sequences evolve at a rate that is relatively constant over time and among different organisms
• Mutations are the raw materials of evolution. Diversity progresses because random mutations occurring at a regular rate

Question 16

State and describe 6 factors which affect the speed of the molecular clock? (PART 1)

Answer 16

1. Bacterial replication rate: A high division rate provides a higher mutation rate
2. DNA or RNA polymerase proof reading fidelity (error rate): Some species (eg HIV) have low fidelity promoting high mutation rate.
3. Selection pressure from the host or environment: High selection pressure removes ‘weak’ mutants and emphasises clusters. Loss of selection pressure allows deletions

Question 17

State and describe 6 factors which affect the speed of the molecular clock? (PART 2)

Answer 17

4. Degree of redundancy (degree of not being useful) in the genome: 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
5. Transmission rate: High transmission rates relative to the mutation rate results in dissemination and single strain outbreaks. (Flu A = 2-3 bases per year and 1.5 transmissions per infection)

Question 18

What factor affects the resolution in sequencing dendograms?

Answer 18

High sequence mutation rate may not affect pathogenicity (Antigenic drift)

Question 19

Between hyper-vairable and conserved gene, what changes more and state the effect of these changes?

Answer 19

• Hyper-variable genes change more rapidly than conserved genes but Conserved genes are more likely to be associated with phenotype and virulence
• Not all changes are new - Some may revert BACK to an older profile (convergent evolution)
• Large and rapid changes are rare but often lead to escape from existing herd protection

Question 20

What is antigenic shift?

Answer 20

• 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 21

Describe the 3 factors and methods involved in epidemological association?

Answer 21

1. Transmission: Hospital acquired infection
2. Reservoirs of infection: Contact tracing - Molecular typing can aid here. Determining Introduction Events - Monitors variants
3. Spread or emergence of resistance: Typically are drug resistant polymorphisms

Question 22

What method can be used to monitor the effectiveness of control measures and briefly describe the process?

Answer 22

• Molecular restriction digest typing
• Process: DNA extracted and cut with the same restriction enzymes then pieces separated on a gel then visualised