Use of Genotyping in Surveillance and Outbreak Investigation

Question 1

What is molecular epidemiology?

Answer 1

Epidemiology is the branch of medical science that investigates all the factors that determine the presence or absence of diseases and disorders.

Molecular epidemiology: “The use of molecular typing methods for infectious agents in the study of the distribution, dynamics, and determinants of health and disease in human populations”

Question 2

What is an endemic disease?

Answer 2

An endemic disease is constantly present in a certain population or region, with relatively low spread (or there may be periods when it doesnt affect people at all, if it is only present in the environment)

Question 3

What is an epidemic disease?

Answer 3

An epidemic disease is when there is a sudden increase in cases spreading through a large population like a country (an outbreak is similar, but usually covers a smaller geographic area)

Question 4

What is a pandemic disease?

Answer 4

A pandemic disease is when there is a sudden increase in cases spreading through several countries, continents, or the whole world

Question 5

What is prevalence?

Answer 5

The total proportion of cases of a disease in a defined population at a given time.
A measure of how widespread a disease is in the population .
Prevalence is influenced by the duration of the disease.

Question 6

What is incidence?

Answer 6

The number of new cases in a defined population during a specified period of time.
A measure of the risk of contracting a disease.

Question 7

What defines an outbreak of an infectious disease?

Answer 7

An occurrence of disease greater than would normally be expected at a particular point in time and in a defined place / population.

Question 8

What is an isolate?

Answer 8

A population of microbial cells from a pure culture derived from a single colony on culture plate

Question 9

What is a strain?

Answer 9

An isolate or group of isolates exhibiting phenotypic and/or genotypic traits that is distinctive from other isolates of the same species

Question 10

What is a subtype?

Answer 10

A strain displaying a specific pattern or set of markers when analyzed by a particular typing method

Question 11

Name four methods for genotyping.

Answer 11

We do genotyping to check if two strains are identical or not.
1) Pulsed field gel electrophoresis (PFGE)
2) Multilocus sequence typing (MLST)
3) Variable number of tandem repeats (VNTR)
4) Whole genome sequence analysis

Question 12

What is typability?

Answer 12

If the method does give unambiguous results

Question 13

What is reproducibility?

Answer 13

The ability of a subtyping method to produce the same result when the analysis on the same microorganism is repeated

Question 14

What is the discriminatory power?

Answer 14

The discriminatory power is the ability of the subtyping method to discriminate between epidemiologically unrelated strains of a species

Question 15

What is MLST?

Answer 15

MLST is the sequencing of a number of “housekeeping” genes (commonly 6-8 genes).
Normally about 450-500 bp length of each sequence.
Any variation in a nucleotide sequence is assigned a new allele number.
The combination of allele numbers defines the sequence type of the strain.

Question 16

What are the steps in MLST?

Answer 16

1) Isolate DNA
2) PCR
3) Sequencing
4) Allele assignment
5) ST assignment

Question 17

What are the applications of MLST?

Answer 17

Epidemiology: MLST is widely used in epidemiological studies to track the spread of bacterial strains and to identify sources of outbreaks.
Population Genetics: It helps in understanding the population structure and evolutionary relationships of bacterial species.
Public Health: MLST data can be shared globally through online databases, facilitating international collaboration in tracking bacterial pathogens.

Question 18

What is a clonal complex (CC)?

Answer 18

Sequence types (STs) are grouped into clonal complexes by their similarity to a central genetic profile (genotype).

Question 19

What is the principle of Spa-typing?

Answer 19

PCR and sequencing of amplification product from the XR region of the spa-gene from S.aureus.
Each unique repeat is assigned a repeat number.
Each unique sequence of all repeats in a spa-gene is assigned a spa type number.

Question 20

What are the mechanisms for change in spa-type?

Answer 20

1) Duplication of repeats
2) Loss of repeats
3) Point mutations

Question 21

What are the two major modes for acquisition of pathogens in hospitals?

Answer 21

1) From another patient, member of staff, the environment, or shared equipment
2) From emergence of resistance to certain drugs in a susceptible isolate that the patient harbours before admission, which then acquires a resistance determinant and become epidemiologically significant

Question 22

What categories can typing methods be classified into?

Answer 22

1) Amplification-based
2) Fragments-based
3) Sequence-based
4) Genomics-based

Question 23

What methods is included in the amplification-based methods?

Answer 23

Different PCR methods like rep-PCR, and VNTR

Question 24

What is fragment-based methods?

Answer 24

These methods involve digestion of DNA by one or more restriction enzymes and different methods of fragment separation and visualization.
Pulsed-field gel electrophoresis (PFGE) is perhaps the best known and most widely used.

Question 25

What is sequence-based methods?

Answer 25

Sequence typing involves the amplification of specific genes, determination of their sequences and then comparison to an international database in order to generate allelic profiles.
While several typing schemes use one locus, such as spa-typing for Staphylococcus, a multilocus typing scheme (MLST) is the most common application.

Question 26

How would a molecular investigation of a CRE outbreak been performed?

Answer 26

If dealing with a local abrupt CRE outbreak which is clustered in terms of location and time, and if WGS expertise is not readily available at the site and comparison to an established nomenclature is not important at the acute phase of the investigation, in order to decide the curbing and/or containment strategies, either PFGE or rep-PCR is recommended. Both are established typing methods that could assist practitioners in real-time investigations.

Question 27

How would a molecular investigation of an Acinetobacter baumannii outbreak in an intensive care unit (ICU) been performed?

Answer 27

As opposed to CRE, MLST may be more useful than PFGE or rep-PCR to compare A.baumannii isolates over longer periods of time in institutions with endemic basal rates of acquisition. In case of an abrupt outbreak, in a unit with low background endemicity, PFGE or rep-PCR might still be used in order to tailor the appropriate preventive opportunities. As a concept, the transmission dynamics of A.baumannii in many ICUs is so complex that both modes of auquisition (pasient-to-pasient transmission, and emergence of resistance) should be tackled concurrently.

Question 28

How would a molecular investigation of a MRSA outbreak been performed?

Answer 28

In MRSA outbreak investigations, if WGS is not readily available, we recommend using either spa-typing of PFGE, depending on the setup of the outbreak.

Question 29

How would a molecular investigation of a VRE outbreak been performed?

Answer 29

As with CRE, if WGS is not readily available, both PFGE and rep-PCR are suitable alternatives to study local VRE outbreaks. MLST is probably preferable only when the outbreak is prolonged, on top of a certain endemic rate and/or broader investigation with comparison to international cloning information is desirable.

Question 30

What factors need to be considered when choosing between non-WGS typing methods for an outbreak?

Answer 30

1) Availability
2) Turnaround time
3) Experience and local expertise
4) Quality control
5) Financial considerations
6) The specific features and characteristics of the outbreak