Molecular diagnostics V

Question 1

What are three approaches to perform diagnostics in virology?

Answer 1

• Virus culture
• Serology
• Molecular biology

Question 2

What are possible virus culture read-outs? (3)

Answer 2

• Morphology/cytopathogenic effect
• Electron microscopy
• Immunofluorescence staining

Question 3

What are possible serology read-outs in virology to identify recent infection/past exposure?

Answer 3

• IgM = recent infection
• IgG = past exposure

Question 4

AFMAKEN

Question 5

What is the goal of MMIZ molecular diagnostics?

Answer 5

Detection, identification & typing of potentially pathogenic micro-organisms

Question 6

What is typing?

Answer 6

Ability to discriminate between different individuals (=strains) of the same species

Question 7

What is the rationale for typing?

Answer 7

Are we looking at clonal expansion or different individuals of the same species?

Question 8

To what kind of outbreak does clonal expansion points to?

Answer 8

Within the hospital

Question 9

A patient receives antibiotic treatment, but is still infected with exactly the same bacterium after treatment. What is a potential explanation?

Answer 9

Treatment not taken

Question 10

What are the two most important recent molecular fingerprinting methods?

Answer 10

• MLST (multilocus sequence typing)
• WGS (whole genome sequencing)

Question 11

What do old school molecular fingerprinting techniques have issues with? (5)

Answer 11

• Reproducibility
• Exchangeability of data
• Discriminatory power
• Complexity
• Genome coverage

Question 12

What is the principle of conventional MLST?

Answer 12

Look at 7 household genes that are always present/positive in the genome of the bacterium of interest

Question 13

Describe the workflow of conventional MLST (2)

Answer 13

• PCR-amplify and sequence ~ 500bp using Sanger
• Bidirectional sequencing to eliminate potential reading errors

Question 14

AFMAKEN

Question 15

What is mass spectrometry in a nutshell?

Answer 15

Charge -> accelerate -> separate -> measure -> compare

Question 16

Name two applications of mass spectrometry

Answer 16

• Bacterial identification
• Antibiotic resistance screening

Question 17

What is the principle of quadrupole mass spectrometry?

Answer 17

Helical separation -> non-resonant particles are separated from resonant particles

Question 18

Bacteria culture for identification & resistance profiling takes a long time. What is the current strategy?

Answer 18

Quick identification & resistance profiling using MALDI-TOF

Question 19

What is the advantage/disadvantage of MALDI-TOF compared to orbitrap?

Answer 19

Advantage = Far cheaper
Disadvantage = Relatively inaccurate

Question 20

How does colistin resistance occur?

Answer 20

Due to modifications to the lipids in the outer membrane

Question 21

How do you detect colistin resistance?

Answer 21

• PCR/WGS (if target is known)
• Lipidomic profiling of bacterial outer membrane (MALDI)

Question 22

What does lipidomic profiling of bacterial outer membranes allow for?

Answer 22

Detecting shifts in lipid composition that resist the mechanism of colistin

Question 23

What is the mechanism of action of beta-lactams?

Answer 23

Inhibit cell wall synthesis

Question 24

B-lactams can be broken down by B-lactamases. This breakdown consists of which two steps?

Answer 24

• Hydrolysis to open B-lactam ring
• Decarboxylation

Question 25

How can MALDI be used in the context of B-lactams?

Answer 25

B-lactamase activity causes mass shift in antibiotics which can be detected using MALDI

Question 26

What is an advantage of mass spectrometry over PCR/WGS?

Answer 26

Shows that a mechanism is actually expressed

Question 27

Antibiotics: What is a drawback of MALDI?

Answer 27

Can only detect antibiotic resistance in hydrolyzing classes of antibiotics (mass change required)

Question 28

What are the advantages of high-resolution mass spectrometry? (4)

Answer 28

• High resolution
• High speed sequencing
• Very sensitive
• Low throughput