Past Papers 11 Flashcards
Oral station - 15 mins
A midwife calls you to say that a follow-up blood sample from a pregnant lady is clearly HIV negative, whereas the first blood sample taken a week before is HIV-1 positive.
Discuss.
2 possibilities -
patient is truly neg, and first result has a problem
patient is truly pos, and second result has a problem
Check patient records - ensure no patient identifier issues, or name changes
Check positive result + paperwork. Was this positive for 2x antibody tests, plus immunoblot? HIV RNA PCR positive? Any issues with controls that might mean this result is an error. Any other problems with run, that might suggest samples have been swapped with a known HIV positive patient. Does patient have a history of HIV infection?
Check negative result - ensure this was actually negative and not entered incorrectly onto computer. Check controls worked, and there are no issues with assay. Check sample has not been swapped with a patient who is known HIV neg
If no obvious patient sampling/ labelling issue, then retest both samples in parallel to understand if those samples contain virus/ antibody.
If discordant results, request a fresh sample for testing, to ensure it is the correct patient sampled.
Oral station - 15 mins
A terrorist bomb victim sustained a penetrative injury from a sharp piece of bone, which originated from a deceased terrorist.
The Emergency Department team is calling you for advice
Apply first aid
Were they wearing gloves?
Visible blood?
Risk of injury - high as penetrating injury
Source - unknown risk. Without knowing much about source, would presume high risk.
HBV - booster vaccine if last 10 years
HCV - follow up
HIV- PEP
HIV PEP - take baseline bloods- FBC/ UE/ LFT, pregnancy test, and send sample for BBV screening to ensure does not already have HIV
Ask staff member to contact occupational health
Oral station - 15 mins
A 32-year-old male sewerage engineer presents with a 1-day history of fever, abdominal pain, headache, myalgia and drowsiness. He had just completed a 3-month assignment in rural Nigeria and returned to UK two days prior to his onset of illness.
The Emergency Department registrar has called you to
discuss.
Risk of VHF - fever within 21 days of returning from VHF country. Nigeria endemic for Lassa, and has risk exposure as rural and sewage worked
More History - caves, unwell contacts, funerals, accommodation stayed in, rat/ mice exposure. Lassa outbreak information via ProMed. Anti-malarial prophylaxis
More clinical history - any bruising, bleeding, vomiting, diarrhoea
isolate as possible VHF - full PPE. Double glove, visor, FFP3 mask, gown, boots, negative pressure room. Inform IPC
Discuss case with imported fever service - consider transfer to Royal Free
Inform UKHSA
Start contact list form
Blood film for malaria
Bloods - FBC/ UE/ LFT/ Coag/ blood culture
VHF screen - blood
Consider leptospirosis - blood/ urine
Consider LP
What is rotavirus pathogenesis?
VP4 capsid protein binds to villus surface
rotavirus non-structural protien (NSP4) acts as an enterotoxin by causing changes in Na+ channels in cells
necrosis of epithelium - diarrhoea
What are benefits of enteric viruses not having an envelope?
Means can resist alcohol gel
Lipid envelope actually means that nucleocapsid is usually quite vulnerable, so once lipid envelope destroyed, virus is subsequently destroyed
Bleach is used to disinfect following norovirus ward outbreak
What concertation is required?
1000 - 5000 ppm
Enterovirus can cause wide variety of conditions
What are possible presentations?
Enterovirus D68 - acute respiratory outbreaks
enterovirus D70 haemorrhagic conjunctivitis
enterovirus 71 HFM, meningitis
Enterovirus A71 - flaccid paralysis
all can cause myocarditisand gastroenteritis
herpangina
https://www.rcpath.org/static/80d54920-b6e2-45aa-bdb6c34026e99179/Part-2-Virology-sample-questions.pdf
You are provided with the following data for a quantitative CMV DNA assay run by your trainee biomedical scientist.
What are the problems with this PCR run? Specify missing items (6 marks)
- ## no R2 coefficint of determination value
- no note of if IQC controls passed
- no date/ name of person who has performed the run
- no negative control - unclear if sample 2/6 have zero copies because they are truly neg, or because PCR failed. Use water instead of DNA as a negative control to detect contaminants in the reaction and to discriminate background amplification
- no positive control - used known patient sample/ IHC or external quality control
- standard curve analysis requires at least 5 data points. This only has 4
- lowest standard is at 1000 - and this doesn’t even have a measured CT value.
Need standard sample which will range from from limit of detection, to 1000 IU/ml
i.e sample 2 is record as having 0 iu/ml. However, it’s true value could be anywhere between 0 and 1000 - highest standard is at 1million.
Needs standard sample in 10m and 100m, as sample 1 is recorded at 86m iu/ml.
if result is above 1m, then result is recorded as “above range of testing”
https://www.rcpath.org/static/80d54920-b6e2-45aa-bdb6c34026e99179/Part-2-Virology-sample-questions.pdf
You are provided with the following data for a quantitative CMV DNA assay run by your trainee biomedical scientist.
Would you accept this standard curve? Please explain why?
Do not accept
No positive/ negative controls
to create a standard curve need a minimum of 5 data points - our graph only has 4
Also want to repeat standards in triplicate, so you are sures that those values are correct
Would like to know the R2 value - the coefficient of correlation and should be > 0.99 to provide a good confidence within the correlation.
This value lets you see if there is a good linear relationship between the values of each sample. A low value could indicate a poor serial dilution. To avoid this, when making your serial dilutions ensure you pipette accurately, using well-calibrated pipettes, and mix dilutions well.
https://bitesizebio.com/31470/obligate-qpcr-standard-curve/#:~:text=To%20perform%20a%20qPCR%20standard%20curve%2C%20you%20set%20up%20qPCR,a%20proportional%20dose%2Dresponse%20curve.
https://www.rcpath.org/static/80d54920-b6e2-45aa-bdb6c34026e99179/Part-2-Virology-sample-questions.pdf
You are provided with the following data for a quantitative CMV DNA assay run by your trainee biomedical scientist.
can any of the results be reported? Please explain
???
Samples 2, 3, 6 cannot be reported, as there values fall below our threshold for limit of detection. There unclear if truly neg, or just low level
Sample 1 - above threshold for testing. Do not report as cannot give an accurate value?
Sample 4 - could report, as result falls within controls
Sample 5 - could report, as result falls within controls
answers very unclear
https://www.rcpath.org/static/80d54920-b6e2-45aa-bdb6c34026e99179/Part-2-Virology-sample-questions.pdf
You are provided with the following data for a quantitative CMV DNA assay run by your trainee biomedical scientist
What do you recommend your lab staff do next?
Repeat run
Add negative control - Use water instead of DNA as a negative control to detect contaminants in the reaction and to discriminate background amplification
Add positive control - in house control - could be old patient sample of external sample
Add another positive standard - to have 5 minimum targets
Add spike porcine virus as positive spike control
Calculate R2
https://www.rcpath.org/static/80d54920-b6e2-45aa-bdb6c34026e99179/Part-2-Virology-sample-questions.pdf
You are provided with the following data for a quantitative CMV DNA assay run by your trainee biomedical scientist
Is this run valid and is the standard curve acceptable? Please explain. (7 marks)
unclear answer?
needs 5 standards
needs R2 calculation
needs standard deviation calculation. CMV IU/ml value should be within 0.2 SD units. As each diluted sample should be 1/10th of the previous sample
Ct values of internal control (spiked phocine herpes virus) - these need compared to know in house values. Because these Ct values seem quite late for an IHC
now has negative and positive controls
What is R2 value?
The coefficient of determination (R²) measures how well a statistical model predicts an outcome.
The outcome is represented by the model’s dependent variable. The lowest possible value of R² is 0 and the highest possible value is 1.
draw a line for CMV viral loads. This means we can estimate with a detected value, what the true value might be
https://www.rcpath.org/static/80d54920-b6e2-45aa-bdb6c34026e99179/Part-2-Virology-sample-questions.pdf
You are provided with the following data for a quantitative CMV DNA assay run by your trainee biomedical scientist
Lower limit of detection was previously determined to be 100 IU/mL
whereas the lower limit of quantification was 400 IU/mL
Define these two concepts and clarify how these are usually determined.
Explain how can a sample be positive but below the limit of quantification
Limit of detection - The limit of detection LOD is the lowest possible concentration at which the method can detect (but not quantify!) the analyte with certain degree of confidence - e.g 99% of the time it will detect <40 copies. It is also defined as the lowest concentration that can be separated from a background noise with some reliability
Limit of quantification -
the lowest concentration that can be quantified with an
acceptable level of repeatability precision and trueness.
e,g can reliably detect >100 copies 100% of the time
and <100 copies only 90% of the time
If LoD is 40 copies, and LoQ is 100 copies, then a value of 60 copies would be detected, but unqauntifiable
https://www.rcpath.org/static/80d54920-b6e2-45aa-bdb6c34026e99179/Part-2-Virology-sample-questions.pdf
You are provided with the following data for a quantitative CMV DNA assay run by your trainee biomedical scientist
How would report these samples:
Sample1
Sample2
Sample3
Sample1
CMV detected above limit of quantification?
Sample2
CMV not detected
Sample3
CMV detected - <400 iu/ml - result is above limit of detection, but below limit of quantification
https://www.rcpath.org/static/80d54920-b6e2-45aa-bdb6c34026e99179/Part-2-Virology-sample-questions.pdf
You are provided with the following data for a quantitative CMV DNA assay run by your trainee biomedical scientist
How would report these samples:
Sample4
Sample5
Sample 6
Sample4
CMV detected 4987 iu/ml
please compare with previous results
please assess for any evidence of CMV related end organ disease
Contact virology to discuss further
Sample5
CMV detected 86400 iu/ml
please compare with previous results
please assess for any evidence of CMV related end organ disease
Contact virology to discuss further
Sample 6
CMV not detected
CMV in vitreous humour has increased by 1.0 log, 1 week after intravitreal ganciclovir administration.
Name 3 laboratory tests for CMV drug susceptibility testing
Genotypic susceptibility testing - particularly UL54 viral DNA polymerase, and UL97 phosphotransferase gene segments. Only useful if you have homogenous viral population, and have known resistance genes you want to target.
Whole genome sequencing (NGS) - improved ability to detect mixed populations for resistance. Will look for all genes, so can detect novel mutations, as well as common mutations
Phenotypic susceptibility testing - plaque reduction assay. Virus inoculated into monolayers of tissue culture. Increasing concentration of anti-viral added. The drug concentration reducing the number of plaques on control monolayers by 50% is referred to as the inhibitory concentration. Can take 4 weeks
CMV susceptibility testing
Why is NGS better than Sanger sequencing
Advantages of NGS include:
Higher sensitivity to detect low-frequency variants
Faster turnaround time for high sample volumes
Comprehensive genomic coverage
Lower limit of detection
Higher capacity with sample multiplexing
Ability to sequence hundreds to thousands of genes or gene regions simultaneously
CMV in vitreous humour has increased by 1.0 log, 1 week after intravitreal ganciclovir administration.
Resistance testing performed
State the relative analytical sensitivities of genotypic assays by Sanger sequencing and NGS
Analytical sensitivity represents the smallest amount of substance in a sample that can accurately be measured by an assay
NGS - higher analytical sensitivity - will pick up 95-99% mutations
Sanger - lower analytical sensitivity - will pick up 80-90% of mutations
CMV drug resistance
How do you prove that a mutation detected by genotypic assay conveys drug resistance or not?
Patient history - how likely is this. Have they previous history of CMV infection with treatment? History of drug resistance
Databank - can help with genotype interpretation. it may be known what the probability of having a resistance gene, in relation to phenotype
Virological - rising VL on treatment with no adherence/ absorption issues
clinical - Treat and observe - if drug has moderate risk of resistance, it is unclear if resistance will occur at the doses given in human treatment. Therefore if patient not unwell, and not expected clinically to be resistant, can treat and observce
Phenotypic testing - although not usually done
CMV drug resistance
What do you understand by false positive, false negative and discordant results in genotypic assays?
false positive - gene may be detected, but it may not have an effect at a phenotypic level. For example patient may actually respond to ganciclovir, and so positive result does not mean it is resistant. Additionally, it can mean it has partial resistance
false negative- gene initially not detected. However as treatment started, non-resistant strains die, and replaced by resistant population. Follow up genotypic testing will show resistance.
False negative - low viral load can mean that the sensitivity of genotypic is reduced
discordant results - mixed populations may produce discordant results, as some viruses are resistant through common mutations or silent mutations, and others are not. After commencing treatment, population may change.
Discordant may also mean that genotypically it is resistant, but phenotypically patient responds to treatment
How would you treat a patient with drug resistant CMV infection?
Check if can reduce immunosuppression/ treat underlying issue e.g HIV
Check what it is resistant to - genotypic
Check why resistant
- inherited resistance
- Acquired resistance - poor compliance, wrong dosing, poor absorption (colitis)
If oral ganciclovir, switch to high dose IV - may be enough to overcome resistance
Consider class switch - Foscarnet, Cidofovir, Maribavir
Novel treatment - cytotoxic T cell infusion
CMV drug resistance
When checking viral loads, when should CMV drug resistance be suspected?
Antiviral drug resistance should be suspected when CMV viremia (DNAemia or antigenemia) fails to improve (ie, >1 log10 increase in CMV DNA levels in blood or serum) after 2 weeks of appropriately dosed and delivered antiviral therapy
Why should Maribavir and ganciclovir not be administered together?
Maribavir competes with ATP for binding to pUL97.This mechanism of action is novel, because it does not require intracellular phosphorylation and is independent of pUL54.
Similar to deletions in UL97, maribavir severely inhibits CMV growth. Although various in vitro mutations have been shown to confer maribavir resistance by modifying the pUL97 ATP binding site, CMV isolates that are resistant to maribavir remain susceptible to ganciclovir (and vice versa).
However, since maribavir inhibits UL97, it impairs phosphorylation of ganciclovir; in theory, these 2 drugs should not be co-administered.
This potential antagonistic effect seems not to be a concern when combining maribavir and foscarnet
Add values to each grade of CMV resistance by filling the three gaps in this table
Grades of resistance Value for each grade of resistance with reference to control strains
High-grade
Moderate
Low-grade
Insignificant <2x
Genotypic data reported as level of resistance EC50 (half maximal effective concentration).
e.g a 5x increase in ganciclovir concentration is required to reduce viral growth by 50% (EC50)
High-grade >15x
Moderate 5-15x
Low-grade 2-5X
Insignificant <2x
What are rarely used alternative treatments for drug-resistant CMV infection?
CMV IgG
CMV specific T-cell therapy
Leflunomide - inhibit viral protein kinase activity
Artesunate - in vitro activity preventing CMV replication
CMV patient of ganciclovir
Genotypic report
UL97 mutation detected
What would you do?
Genotypic does not necessarily mean phenotypic issue
UL97 mutation confers low level Ganciclovir resistance
Genotypic does not necessarily mean phenotypic issue
Assess clinical disease and viral load
- If VL not increasing, then continue treatment
- Switch oral to IV
- consider increasing dose to max e.g 10mg/kg every 12 hours
- switch drug class
If VL increasing, then switch to alternative drug e,g Foscarnet or Cidofovir
CMV patient of ganciclovir
Genotypic report
UL97 mutation detected
UL54 mutation detected
What would you do?
Genotypic does not necessarily mean phenotypic issue
Assess clinical disease and viral load
- If VL not increasing, then continue treatment
- Switch oral to IV
- consider increasing dose to max e.g 10mg/kg every 12 hours
- switch drug class
If VL increasing, then switch to alternative drug e,g Foscarnet or Cidofovir
How do you prove that a mutation detected by genotypic assay conveys drug
resistance or not? [5 marks]
Database - check drug resistance database
Clinically - e.g worsening colitis/ retinitis
Clinically - rising viral load
Phenotypic testing - plaque reduction assay
