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EBV viral load measurement from the patient to the result in

By Dale Butler,2015-03-07 17:45
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EBV viral load measurement from the patient to the result in

    EBV viral load measurement:

    From patient to interpretation in transplant recipients

    To progress with EBV viral load standardization we ask you today to share your experience about EBV viral load testing in transplant recipients with us. In order to summarize all data we have set up a semi-standardized questionnaire. Please feel free to add any comment you feel that might be important. We do not only ask how you are performing EBV viral load measurement but also why you choose this or that method. Selecting one or the other method might be a result of

    ; theoretical reasons

    ; data from others (e.g. literature, expert opinion)

    ; own experience

    ; other reasons

    We would be very thankful if you could indicate which reason was relevant for you. Moreover, it would be great if you could specify the reasons e.g. if you tested different methods and as a result selected one method it would be excellent if you could give more detailed information about your evaluations. If you feel that this is too time consuming at the moment we would be happy if we could contact you again and ask for details for certain questions.

    Performing viral load measurement in a certain way does not necessarily mean that this is the methods you prefer as a standard. Thus, for some topics we ask separately what you prefer and why.

    We know that you will need some time to answer all theses questions, but we think that this is the only way to combine all our experience about EBV viral load measurement to find a consensus. When we publish the consensus all who answered the questionnaire are co-authors. To help you with filling out this form you find here the questionnaire and the answers of one lab.

Thank you very much for your help!

    Name Barbara Gärtner

    Email barbara.gaertner@uniklinikum-saarland.de

    Address Institut frü Virologie, Haus 47, D-66421 Homburg/Saar, Germany

    Phone ++49-6841-1623950

    Fax ++49-6841-1623950

    1.) Patients

    Could you describe the patients you are testing for EBV viral load (e.g. how many solid organ transplant recipients (which organs), stem cell transplant recipients, adults, children)?

    Solid organs: lung 30%, heart 10%, liver 10%, kidney 10%, stem cell 40% adults 90%, chilrden 10%

    Over all how many EBV PCRs do you perform per week (all EBV PCRs not only the PCRs for transplant recipients?

90

How often (per week) do you perform EBV PCR?

daily

    2.) Sampling

    How often do you test the patients in routine? Are the intervals different according to age of the patients for solid organ recipients or for stem cell transplant recipients? Are the intervals different for adults and for children? On what are the intervals depending?

    Once a week, when patient are in hospital. At each visit in an ambulant setting. Only in very few cases (increase in viral load, clinical signs for PTLD) we test twice a week or even more frequently. This is the same for all transplant recipients and all ages.

Please select a reason why you choose these intervals.

other reasons

Could you please specify your reasons?

    The maximal frequency (normally once weekly) is defined by us, the minimum by the clinician. Thus I have no influence on it. To test as a maximum once weekly is our decision and more a consequence of otherwise to high workload. Moreover, I have no evidence from literature that EBV viral load should be tested more frequently in routine.

    3.) Matrix

    Which matrix do you use for testing other than blood (e.g. CSF, biopsies)?

CSF, biopsies, in rare cases urine and stool

    If you test blood, which matrix do you use (serum, plasma, whole blood, PBMC, B cells a.s.o.)?

mainly whole blood, only in rare cases serum/plasma

Please select a reason why you choose this sample matrix.

theoretical reasons

Could you please specify your reasons?

    Since the proliferating B cell is not in the cell free compartment, I choose a compartment with cells (whole blood) and not serum or plasma.

    In our experience the exact viral load is not as important for the diagnosis as is the dynamic of the viral load increase. Thus, I think the advantage of PBMC or B cells reflecting even better the proliferating B cell than whole blood is not compensated by the higher work load.

What would you prefer for a standard material?

    I am not sure whether whole blood or plasma is better. It might be the case that both works. We need a study with a clinical endpoint to evalute this question. Unfortunately I never compared both with a clinical endpoint (I only compared the viral loads in both compartments and found that whole blood had higher titers and was earlier positive compared to plasma but I have no data showing an effect on treatment and outcome in the patients. It might be the case that whole blood is more sensitive and plasma is more specific and in the end both are suitable.

    4.) Extraction

Which method do you use for extraction?

Magnetic beads (EasyMAG)

Please select a reason why you choose this extraction method.

own experience

Could you please specify your reasons?

    We tested different extraction methods, silica extraction (Qiagen), and different magnetic beads (MagaPur compact, Qiagen) and the extraction using Easy Mag was even better than silica by hand (this applies only for EBV) and by far better that the other two mangentic bead extractions.

    Do you think we should standardize the extraction? If yes, what would you prefer?

    No, I think the method for extraction must be defined by the lab since it is not feasable to have different methods in a lab. Thus the method of extraction might by choosen due to different other requirements of the lab since most lab do not only perform EBV viral load.

    5.) PCR-technique

    a. Technique

Which PCR technique are you using?

LightCycler with Hybprobes

Please select why you choose this method?

own experience

Could you please specify your reasons?

    We tested earlier quantitative competitive PCR with detection on a gel. However this was too time consuming. Interestingly, the evaluation of both methods showed only a weak correlation. The same was found by others as well (e.g. Stevens). However, since there is no goldstandard this evaluation is difficult to interprete.

    b. Location of primer and probes

Where in the EBV genome are your primers and probes located?

    within p23 (VCA) see Ibrahim et al. J Clin Virol. 2005 Jan;32(1):29-32

Please select why you choose this location?

data from others (literature, expert opinion)

Could you please specify your reasons?

    We adopted the PCR (including the plasmid) first from another group (Schwarzmann, Regenburg). Later when we changed to LightCycler PCR we changed the primers slightly.

What genome location would you prefer as a standard?

    I have no perference except that the regions in the genome with repeat sequences must not be used. These regions are excellent for a highly sensitive qualitative PCR but suboptimal for quantification. Since we do not need a high sensitivity (except for testing CSF) these regions must not be used.

    c. Length of fragment

How long is your PCR fragment?

355 bp

    d. Method of quantification

Which method do you use for quantification?

external standard curve

Please select why you choose this method?

other reasons

Could you please specify your reasons?

Its was the easiest for us (easier than internal standards).

What would you prefer as method for quantification?

I have no preference

    e. Standards

    What are you using as a standard for quantification in your assay (e.g. plasmid, Namalwa, diluted in what?

plasmid diluted in a background of EBV negative DNA

Please select why you choose these standards.

other reasons

Could you please specify your reasons?

    It was the easiest for us, since our coworker provided the plasmid. When we established the PCR we correlated our plasmid standard with Namalwa cells to show that our calculations were correct.

    First we used plasmid DNA in buffer. However, the curves of the plasmid on the LightCycler were different compared to the curves of the patients showing a sharper increase in standards. Since we are using the 2nd derivative method for quantification, the shape of the curve is relevant for the copy number. When diluting the plasmid in DNA from whole blood of a EBV negative individual the curves of plasmid standards and patients increase in parallel. .

What do you prefer as a standard and why?

    Namalwa cells might be best for a standard, since they can be quantified by counting and might be used in all PCR setting.

    f. Inhibition control

Do you use inhibition controls, if yes, which controls?

external inhibition control by spiking the sample with EBV DNA

Please select why you choose this method.

theoretical reasons

Could you please specify your reasons?

    For us it was the easiest although the most expensive since we have to perform a second PCR for each sample.

    g. Unit

    In which units do you report your results, if you use more than one unit please indicate.

copies/ml (in few cases in addition copies(µg DNA)

Please select why you choose this units.

own experience

Could you please specify your reasons?

    We earlier tested copies/µg DNA and later changed to copies/ml. When I compared our results in many many sample the increases and decreases were almost the same no matter if copies/ml or copies/µg DNA were choosen. Theoretically one would assume that copies/µg DNA is a more exact measurement in samples with cells, however, in practical the cellular DNA does not fluctuate as much as the viral load does.

Which units do you prefer and why?

    I would prefer copies/ml (or even better international units/ml) since it is easy and we have no data with a clinical endpoint showing that other units are superior.

    6.) Interpretation

    For which aims are you using viral load detection in transplant recipients? (Please tick the box, you can tick more than one box).

     diagnosis of PTLD

     preemptive therapy

     to guide immunosuppression

     other reasons

    a. Cut-off or dynamic

    Did you establish a cut-off for one these aims, if yes please indicate the cut-off in the table? If in addition (or only) the dynamic of the increase in viral load is relevant please indicate in the table the increase in log (during which time) which is relevant for your decision.

    Aim Cut-off Dynamic (in log)

    Diagnosis of PTLD

    Preemptive therapy

    Immunosuppression

    other

If the table does not fit to your decision tree please explain?

    We have no clear cut-offs even not for the dynamic. A 2 log increase within a week or a viral load of more than 100.000 copies/ml was in many patients with reactivation suggestive of a PTLD or at least a reason for preemptive therapy. In patients with primary infection after transplantation a viral load of 100.000 copies/ml or even more means often nothing. When we see viral loads of 10.000 copies/ml we call the clinical and discuss together what to do.

    b. Which other methods (EBV detection) are relevant for you when

    interpreting the viral load (e.g. EBV specific T cells, EBV mRNA?)

    In some patients we test EBV viral load in cells and in plasma to get an impression about the amount of lytic or latent replication (although I am fully aware that this method is to exact for this purpose).

    c. Which other factors (e.g. age of patients, time after transplantion

    a.s.o.) are important for you in interpretation of a viral load?

    The three factors which are most important for me are 1.) the time after tranplantation, 2.) the EBV status before transplantation (primary infcetion reactivation), 2.) the immunosupression. The age is not so important for me.

    7.) Comments

Do you have any comments?

    I think we should keep in mind that a standard working for all should most importantly be easy even if it is not 100% exact.

    I would be interested to learn more about the difference between primary infection and reactivation. I have the feeling that we need different cut-offs for both.

    Many thanks for your help

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