Questions from S 8 Flashcards
Actual exam question
Chief of Pathology services asks for your opinion on which sequencing platform would be better suited to your hospital’s diagnostic needs - Nanopore or Illumina for sequencing
Currently you outsource all your sequencing to private or UKHSA laboratories
Outline how would you answer this question
Why are you sequencing - e.g diagnostic v research
Do you have lab space/ budget
Do any staff have previous experience on either platform
Nanopore
- 87-98% accurate
- - faster TAT
- short or long read. Long read useful for looking for novel viruses or new mutations. Typically better for research
- portable machine
Illumina
- more accurate reads >99.9%. Better for clinical diagnostics as accuracy is most important
- short read only - useful for known viruses and to focus reads on areas on known viral variation. typically better for clinical diagnostics
- requires laboratory setting
Nanopore and Illumina are both methods of NGS
How do their methods differ?
Illumina -
Sequencing by synthesis (ATCG)
- DNA chopped up into smaller segments.
- Then attached to solid phase flow cell surface.
- These DNA strands them amplified into millions of clusters of same DNA segments
- Then sequencing by synthesis occurs whereby fluorscently labelled nucleotides are added to complete the DNA segments
- Fluoresence is analysed to determinte what the nucleotide sequence must be
Nanpore -
A nanopore is a tiny hole (a nanometer in size) formed by the structural conformation of a protein. Each DNA or RNA molecule that passes through one of the nanopores disrupts the current in a different way, enabling the identification of the molecule and its DNA or RNA sequence.
Nanopore and Illumina are both methods of NGS
The methods of NGS are different.
What are the benefits of either method?
Nanopore - long-read sequences
Illumina - short-read sequences
Short and long read lengths are best for different applications. Short-read lengths are best for applications like small RNA sequencing and gene expression profiling. On the other hand, long-read lengths are best for de novo assembly because they allow for more sequence overlap.
Illumina and Oxford Nanopore tend to be associated with different read lengths. Although Illumina’s product offering has different sequencers for short-read and long-read sequencing, it is best known for short-read sequencing products. By contrast, Oxford Nanopore is best known for long-read sequencing. In fact, nanopore sequencing provides the longest read lengths of all NGS technologies
What are the uses of whole genome sequencing?
Classification/taxonomy of viruses
Identify novel pathogens
Identify mutant variants/ resistant strains
Outbreak investigation
Study the human microbiome
A research centre donates a Nanopore minion, flow cells, and reagents. Your scientist is willing to attempt to sequence some samples with known pathogens. They have collected these samples in the lab freezer.
What advice and considerations should you have for the use of these samples?
Training - is the scientist appropriately trained to perform testing?
Freeze/ thaw cycles - reduced quality of sample could affect results
Ensure correct sample types for testing - e.g blood/ CSF
Need to send to reference lab for sequencing, to confirm your sequencing is correct
Risk of identifying another pathogen, which may affect patient management.
Guidelines - RCPath - Guidance on the use of clinical samples for a range of purposes that are not within the remit of Research Ethics Committees
A research centre donates a Nanopore minion, flow cells, and reagents. Your scientist is willing to attempt to sequence some samples with known pathogens. They have collected these samples in the lab freezer.
You identify an incidental finding on new testing.
What needs to be done?
Explanations
- new test is better sensitivity/ specificity
- sample degradation in freezer
- error during previous testing
- error during current testing- lack of training/ SOP
Guidelines - RCPath - Guidance on the use of clinical samples for a range of purposes that are not within the remit of Research Ethics Committees
If samples cannot be un-anonymised, then you cannot do anything
If can identify original patient:
- if not clinically significant, than can ignore
- if would affect patient care, have a duty to inform patient/ patient team
- if patient has died, escalate to identify whether this may be a possible cause of death
Actual exam question
Working group has asked you to write draft justification for introduction of HCV screen in Infectious Diseases in Pregnancy programme for 2023.
Write the first draft of your report to cover your existing antenatal program and benefits, problems, and costs associated with introducing HCV testing
Focus on benefits here
Benefits are you already have clot sample, so can just add on anti-HCV testing easily
Benefits can do testing on hard to reach groups - sex workers/ IVDU
Could do targeted testing - e.g just IVDU/ sex worker
Establish true prevalence of HCV
Get more people diagnosed, more people treated, and potentially eliminate disease
Actual exam question
Working group has asked you to write draft justification for introduction of HCV screen in Infectious Diseases in Pregnancy programme for 2023.
Write the first draft of your report to cover your existing antenatal program and benefits, problems, and costs associated with introducing HCV testing
Focus on why it currently is not recommended
Screening is not recommended in pregnant women. This is because it is not known:
how many pregnant women in the UK have hepatitis C
why some mothers pass the virus to their child and others don’t
how accurate screening tests are for hepatitis C in pregnant women
how effective treatments for hepatitis C would be for pregnant women and their children
if treatments would prevent unborn babies from catching hepatitis from their mother
cost - serology/ PCR/ genotyping/ hepatology and treatment. Especially if patient might clear themselves
Low prevalence, means high risk of false-positive result. Each positive needs confirmation/ RNA testing
Pregnant lady with HIV infection
When might this patient be considered low or very low risk?
Very low risk
viral load at booking <50 copies/ml
been on ART for >10 weeks
2x viral loads <50 copies/ ml, 4 weeks apart
viral load at delivery <50 copies/ml
Low risk
viral load at booking <50 copies/ml
been on ART for >10 weeks
2x viral loads <50 copies/ ml, 4 weeks apart
If viral load at any point goes above 50 copies/ml
Pregnant lady with HIV infection
When might this patient be considered high risk?
High risk
Viral load >50 copies/ml at delivery
If viral load for example is 1000 copies at some point during pregnancy, but was <50 on day of delivery, this patient could be reduced from high risk to low risk
Pregnant lady with HIV infection
What is neonatal treatment options for these risk categories?
Very low risk
Low risk
High risk
Very low risk
2 weeks zidovudine
Low risk
4 weeks zidovudine
High risk
4 weeks zidovudine/ Lamivudine/ Nevirapine
Pregnant lady with HIV infection
What drugs are available IV for neonate?
Zidovudine only IV preparation
Enfuvirtide can be given subcutaneously. Some use in drug resistant infection. But unlicensed as its pharmacokinetics are unclear
Pregnant lady with HIV infection
How soon should PEP be started for neonate?
within 4 hours of delivery
Pregnant lady with HIV infection, known to be resistant to zidovudine.
High risk neonate
How does this affect neonatal management?
Still give zidovudine monotherapy
Resistance may not be 100%
Even if virus is 100% resistant, it is likely less fit, and therefore transmission of zidovudine resistant HIV virus is less likely to occur, as wild type virus is more likely to transmit
Zidovudine resistance has not been shown to increase risk of HIV transmission in neonates
Pregnant lady with HIV2 infection
High risk neonate
How does the management change?
Nevirapine is not effective in HIV2 - NNRTI
Trial zidovudine/ lamivudine/ raltegravir until guidance is available
