Michi Huber - Bloody Diagnostics

Question 1

<p>Give 4 direct methods to detect viruses</p>

Answer 1

<p>Antigen detection
<br></br>Genome detection
<br></br>Electronic Microscopy
<br></br>Isolation of Virus</p>

Question 2

<p>Give 2 methods for indirect detect of viruses.</p>

Answer 2

<p>- Ab detection
<br></br>- CPE detection</p>

Question 3

What’s the difference between an Antigen and Antibody Elisa ?

Answer 3

Antigen elisa: Ag is capture by fixed ab on plate, then detected using a labeled secondary.
Antibody elisa: Ag is fixed on the plate, then detected with primary and labeled with secondary.

Question 4

What’s the three ways you can detect viral NA ?

Answer 4

Hybridization techniques (binding of labeled probes)
Amplifications by PCR
Sequencing.

Question 5

What’s a Nested PCR ?

Answer 5

Two successful rounds of PCR; with primers for the second designed to bind within first round product. Increases specificity.

Question 6

What’s the purpose of a viral enzyme assay ?

Answer 6

Measuring the activity of such and such enzyme of the virus (e.g. RT) to get a measurement on the infection or diagnostic.

Question 7

Which are the three methods to isolate and measure viral replication?

Answer 7

Embryonated eggs (IAV)
Animal culture (In Vivo)
Cell Culture (In vitro).

Question 8

What’s the drawback of cultivating the virus on cell lines ?

Answer 8

Might lead to weird adaptations,

Question 9

What’s an end point dilution method ?

Answer 9

Dilute the virus, infect cells with lots of replicates for each dilution, then check by ELISA where the antigens are detected. You can then calculate the TCID 50 from that. It’s not super precise, but you have formulas to avoid that problem.

Question 10

What’s virus neutralisation and how can you measure it ?

Answer 10

Neutralisation is the inhibition of viral infectivity by antibody or drug activity.
It can be measured by addind Abs or drugs to a virus preparation and seeing how it gets affected.

Question 11

What’s neutralisation assays useful for, in a diagnostic sens ?

Answer 11

It can identify serotypes.

Question 12

What’s the purpose of sequencing viruses ?

Answer 12

genotyping them
Phylogenetic analysis
Detection of drug resistant mutants
Detection and identification of new viruses or variants.

Question 13

What’s the main difference between Sanger and NGS ?

Answer 13

Sanger is doing bulk sequencing in a single tube,
While NGS is sequencing of individual molecules through chips, resulting in millions of sequences. It’s also muuuch higher throughput, as Sanger does 1 million bases per day when NGS does 50 gigabases per day.

Question 14

What’s the nanopore sequencing principle ?

Answer 14

It’s a 3rd gen. sequencing method where the DNA strand goes through a nanopore, resulting in small ionic current changes as different bases go through. There’s no optical readout but it’s awesoooome.

Question 15

When is Metagenomics a good tool?

Answer 15

Up to 60% of cases of respiratory infections, gastro and meningitis are from unknown etiology: boom metagenomics is there to detect what could be the cause.

Question 16

Why is it quite important to diagnose viral pathogens ?

Answer 16

Although antivirals are rarely available, it’s still important to isolate patients, avoid unnecessary Antibiotic use, and reduce costs due to useless treatments or additional diagnostics.

Question 17

Pros and cons of Metagenomics compared to other stuff ?

Answer 17

It’s pan-pathogen detection in a single reaction, but it’s quite expensive and slow.

Question 18

What’s the metagenomics workflow ?

Answer 18

Total NA are extracted from the patient sample, then RT is performed to get cDNA from the RNA + the DNA already extracted. This library is then prepared and completely sequenced. The results are then processed through the virMet bioinformatics pipeline to link sequences to genus.

Question 19

What’s the clinical impact of metagenomics ?

Answer 19

Benefits complement routine tests in challenging diagnostic scenarios.
Gives an unbiased detection of viruses usually not detected in routine diagnostics.
A single test replaces multiple.
Provides a lot more information as you get the full genome and genotype of the identified pathogen.

Question 20

What’s the few challenges remaining for metagenomics, and what would be the ideal scenario ?

Answer 20

It’s still quite slow, quite expensive and demanding, and not quite as sensitive as we’d like it to be. contaminations can also be quite the problem. And it’s difficult to differentiate between commensals and pathogenic organisms.
The best case scenario would be one test to fit all organisms and do a rapid and cheap sequencing.

Question 21

Another use: studying interplay in intestine linked with diagnostics

Answer 21

HIV-associated AIDS with alterations in microbiome due to AdV.