Molecular Markers I

Question 1

What are the typical steps of a molecular epidemiology study design?

Answer 1

Study objective (question you’re trying to answer, hypothesis you’re addressing)
->
Sample choice (time interval, setting, organism. At which level do you want to type)
->
Obtain isolates (sampling, DNA extraction, culturing strains)
->
Select molecular marker (convenient, stable, discriminatory power)
->
Select typing method
->
Analysis (organise data, arrange by similarities and differences)

For the last 3, one has in mind the organism, typeability, reproduceability, discriminatory power, ease of interpretation and ease of use.

Question 2

Can you list some applications of molecular epidemiology?

Answer 2

“classical” epidemiology
- outbreak investigation
- transmission system
- surveillance
- Identify new pathogens

“Derived” (secondary) applications
- pathogen origin and evolution
- host-pathogen coevolution

Question 3

Molecular epidemiology can help identify…

Answer 3

• the microorganism responsible for the disease
• the source
• route of transmission
• genetic relatedness of isolates to each other
• genes related to virulence and drug resistance

Question 4

Sampling sources and steps include…

Answer 4

• Patient isolates from hospital.
• Clinical specimens from Outbreak.
• Food items collected during outbreak investigations.
• Environmental samples (water,soil..)

culture isolate (if possible) -> nucleic acid or protein extraction -> typing (choose molecular marker of interest -> typing method -> analysis)

Question 5

What are the two categories of markers on can use for a molepi and give an example of each.

Answer 5

Phenotypic markers (“old fashioned” conventional typing methods):

Biotyping, serotyping, bacteriophage typing, antimicrobial resistance etc.

Molecular markers: based on the physical characterization of bio-molecules
(nucleic acids, proteins, fatty acids).

Can also do strain typing.

GENOTYPING is the most common
nucleic acid based-typing methods.
-SNPs (Single Nucleotide
Polymorphisms)
-Insertions
-Deletions
-Transposons (Insertion Sequences)
-VNTRs (Variable Number of Tandem
Repeats)
-CRISPRs region (Clustered Regularly
Interspaced Short Palindromic Repeats)

Question 6

When selecting an appropriate marker, what are three types of criteria you should keep in mind?

Answer 6

Convenience
Appropriateness (Discriminatory power and stability)

Question 7

A “convenient” marker has the following characteristics…

Answer 7

• “Typeability”: Ability of the technique to generate an unambiguous result for an
  isolate tested.
• Simplicity: Ease of performance, less training, accessible to more laboratories
• High throughput: Process a large number of specimens simultaneously
• Reproducibility: Or reliability, is the ability of a test to produce identical results when a strain is tested repeatedly.
• Ease of interpretation: Amenability to computerized analysis and incorporation
  of typing results into an electronic database.
• Cost and Speed

Question 8

How could you increase the discriminatory power of a typing method?

Answer 8

The higher the number of discrete units of typing
information generated by a typing method from a set of
isolates, the higher the discriminatory power of that test.

↑ Types = ↑ Discriminatory power

Looking at just one locus, may make distinct species look more similar than they are.

The ideal, in terms of power, is to choose something that will give you the highest amount of info with the lowest cost whilst still allowing you to differentiate between and compare your chosen isolates.

(1 gene kids and one ice cream vs 3 genes from kids and 6 ice cream parlours)

Question 9

Why is stability of a marker important?

Answer 9

Different areas of the genome change at different rates and this differs between organisms (molecular clock).

HIV very quickly, Mtb not so.
As you are aiming to compare isolates within a molepi, too much or too little difference can make inferences hard. You want to correctly conclude the truly unrelated strains as different and the truly linked strains as similar.
For HIV, you’d need to look at a whole genome to make comparisons whereas for Mtb you could look at a couple of genes.

 The duration of stability of the typing information will determine the
applicability of the marker for epidemiological purpose:
-Stability of genetic marker for 6 months = Outbreak
investigation.
-Stability of genetic marker for 2 years = Surveillance
purpose of multiregional comparison of strains

When telling strains apart, you may only need to use one gene it is evolves fast enough to accrue differences.

Question 10

Important descriptors/ parts of a dendogram?

Answer 10

Root
Trunk
Node
Branch
Branch length
Leaf nodes/ tips (labels at end of tree - extant strains upon which tree is based)

When comparing trees, the topology is the tree structure, the pattern of relatedness. This will be the same with a cladogram and phylogenetic tree, however, when genetic distance is included in the phylogenetic tree, the topology may remain the same, but the trees wouldn’t be equivalent.