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Dr Singh Talks on her CFS XMRV study and the WPI's Response

Discussion in 'XMRV Research and Replication Studies' started by Cort, May 17, 2011.

  1. Cort

    Cort Phoenix Rising Founder

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    The Human Isolate Question-The WPI appears to propose that their use of a human isolate of XMRV allowed them to find an XMRV-like family of viruses containing VP62 strains, p variants of the virus (Lo et. als pMLVs) and other strains and that Dr. Singhs use of an XMRV clone developed from the VP62 strain lead her to miss those strains and that is one reason that she didnt find anything.


    (Note though that the WPI did say that the patients they directed to Dr. Singh tested positive for antibodies to the VP62 strain - which, given the increased sensitivity of Dr. Singhs tests she should have been more effective at picking up.The WPIs argument regarding using a human isolate possibly explain reduced results but its hard to understand how it could explain zero results. The data from the original study indicated that tests using VP62 were adequate to pick the type of XMRV found in that study. ).


  2. Cort

    Cort Phoenix Rising Founder

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    Dr. Satterfield of Cooperative Diagnostics on Dr. Singhs XMRV CFS Statement

    Dr. Satterfield of Cooperative Diagnostics explains about the dUTP_UNG system and provides his take on Dr. Singhs comments on XMRV..

  3. Megan

    Megan Senior Member

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    Cort,

    Thanks for your interview. I accept that there is weighty evidence is against XMRV now, but it still seems to me there are outstanding questions regarding both XMRV and other possible virusus/retroviruses. One of these is in relation to what were the positive antibody tests in referred to in Singh's patent document picking up? The relevant quotes from the patent are below. I understand both of these tests to be antibody tests (ie not subject to contamination). I suppose she would say they are cross reacting to something else, but the question remains what? especially if the tests are negative in controls as stated. Surely this is an important question that we CFS patients ought not let go?


    Quotes from patent document:

    [0027] Figure 13 shows Western blot detecting presence of antibodies against XMRV polypeptides. A serum sample from a patient with chronic fatigue syndrome shows antibodies against SU, CA and pl5E polypeptides (A). In (B) is a negative control serum, which does not show reactivity to any of the XMRV polypeptide.

    [0134] Similarly, to detect if patients with Chronic Fatigue Syndrome have evidence of XMRV infection, we collected sera from 105 patients diagnosed as having chronic fatigue syndrome and fulfilling the Fukuda criteria for diagnosis. For comparison, we also collected sera from 200 healthy volunteers. Each of these sera was used to probe a PVDF membrane onto which XMRV proteins had been transferred from a gel. A positive sample usually contained antibodies reactive to at least two of the following three XMRV polypeptides: SU, CA and pi 5E (see Fig. 13A). Non-reactive samples (most healthy volunteers) did not contain antibodies to SU or pl5E (Fig. 13B). Antibodies to CA alone, however, were quite prevalent in the healthy volunteer population, and are not to be interpreted as evidence of XMRV infection.
  4. Ernie

    Ernie Senior Member

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    What's this look who's presenting at Cold Spring Harbor Retrovirolgy conference on May 23, 2011. I guess she did find it in prostate cancer with a different assay.

    Singh, I.R. XMRV, or a related virus, is present in a large percentage of men and is localized to the androgen secreting Leydig cells of the testis
  5. August59

    August59 Daughters High School Graduation

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    I wonder if she will pull her poster now? Cozak, Sarafianos and Paprotka will be represented at this conference as well by either a poster or talk. Could be interesting!

    http://meetings.cshl.edu/meetings/retro11.shtml
  6. Ernie

    Ernie Senior Member

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    She is not pulling it.
  7. eric_s

    eric_s Senior Member

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    Will be interesting to see what method she used to find XMRV. I guess she looked somewhere else than in the blood, as she found nothing there in the ME/CFS study, but if she says it's found in a large percentage of men, it can't be prostate tissue either, i guess.

    A large percentage sounds like something higher than ~4%. Interesting... maybe something we've never heard before.

    Edit: Oh, ok, the text probably answers my question already. But what lead her to look there? Maybe her autopsy study?
  8. Cort

    Cort Phoenix Rising Founder

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    That's an amazing statement...XMRV or a 'related virus' and its in the testes....that's got to be the autopsy studies....

    One question is is it doing anything? If it's the autopsy study she should be able to determine if XMRV's presence is associated with illnesses. If nobody had any physical problems that would seem to indicate testical problems then she will probably scratch XMRV off the 'damage forming pathogen' list.

    It would be disappointing if that was the only place she found it..

    Of course that statement does indicate that XMRV is a virus that infects humans - which she has always believed.

    It could end up working out both ways....XMRV has escaped from the lab and contaminated samples via the 22RV1 cell line and it is a virus that is, out in the wild so to speak, and is infecting humans. Its even possible that the WPI has examples of both! That the 22RV1 cell did get into Silverman's samples and hence to some of the WPI samples and they also picked up the wild virus as well in their sampling. :)

    Silverman's paper will be so key....has he been able to uncover contamination or has he been able to discount that. Can he now prove that his XMRV integration sites reflect human infection and not contamination (as an earlier paper suggested) or do they reflect contamination?

  9. Gemini

    Gemini Senior Member

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    Both her blood and tissue results are consistent with Emory's macaque findings
    where XMRV disappeared from blood rather quickly and was later found in organs...
  10. August59

    August59 Daughters High School Graduation

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    The website for the conference states that all of the abstracts had to be submitted by March the 8th. That is a few weeks back! That is why I was asking about her pulling it, but I imagine her study was already completed by then anyway. Dr. Cosak has been very quite lately too, so I curious to see what she brings to the conference.
  11. heapsreal

    heapsreal iherb 10% discount code OPA989,

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    she will have to prove its not contamination????
  12. Cort

    Cort Phoenix Rising Founder

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    I think my statement was misinterpretated by someone to suggest that I believe this finding rescues XMRV from the plight it is in with CFS. I don't. I think XMRV's connection to CFS rests in labs finding it in the blood of people with CFS - not in their tissues. My personal opinion is that - after a year and a half of searching for it - that that's not going to happen. The most likely scenario IMHO is that Silverman will announce at the conference that the 22RV1 cell line contaminated his prostate samples thus probably providing a smoking gun pointing at the WPI.

    On the other hand Silverman could say that the opposite; that his prostate findings are real and that 22RV1 did not contaminate his samples. Since there is new evidence pointing to XMRV being an genetically distinct infectious virus - I think its possible that Dr. Mikovits picked up some of that if she is using tests that search for an array of viruses. She did not, to my knowledge do that with the Lombardi paper - she simply looked for the gag and env genes from prostate cancer samples - and they there were in spades. I think the search for more genetic variability bypasses the fact it wasn't necessary in the original paper.

    We'll see where the science takes us.

    This is really difficult stuff and if this is how it all turns out - no one is to blame. These are obviously incredibly difficult subjects which good labs have gotten wrong before - and Silverman's and Dr Ruscetti's and by implication the Dr. Mikovit's are obviously some of the best.

    I imagine that, however, XMRV turns out - the researchers will be pointing to it and thinking about it for a long time. The possibility that it was created in a lab has got to really send some minds turning.
  13. acer2000

    acer2000 Senior Member

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    Yep... and still nobody has proposed a study on tissues in humans with CFS. I know blood is easier to test, and I know some people have found it there, but come on... at this point its pretty obvious based on the monkey study someone needs to do tissues.

    The CDC study with prostate cancer also is consistent. Switzer also found it in prostate, but not in the blood.

    That and the following need to be addressed:

    - Differences in positive rates between patients and controls in the non 0/0 studies. If contamination is responsible, how can the results line up so neatly in favor of patient positives when both sets of samples were run through the same reagents/cell lines/etc.. There may be a good reason for this, but it needs to be figured out.

    - Nobody has "exactly" replicated the methods from the original paper. It doesn't matter that other labs think they might have a different/better process. At this point, someone needs to go back to square one and do an exact replication. Any other method just adds to the confusion. Details matter. We have to crawl before we walk. And we have to walk before we run.

    - Some patients who have taken ARVs (such as Dr. Jones, and Dr. Snyderman) are clearly doing better and (at least in the case with Dr. Snyderman) his immune parameters and cancer markers are improving. This needs to be explained. If not XMRV, what are these ARVs treating? Do they have NK cell counts as well as immune markers from various points on treatment?

    - The recent paper from the WPI regarding the immune parameters that correlates 95% with the XMRV positivity. I'd like to see them go further and correlate it with NK cell function.

    - The antibody tests need further work. Why are some people PCR/culture positive but antibody negative? Why is the opposite sometimes true? If not XMRV they are picking up, what are they picking up?

    Also - we know from the monkey study that immune stimulation draws XMRV back out into the blood. We also know from experience that exercise exacerbates the symptoms of ME/CFS. Why not do a study where you test from XMRV (using the proven methods) following either of these events? Would that not increase the theoretical likelihood of finding it?

    Well.. that's what I'd do. :)
  14. eric_s

    eric_s Senior Member

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    I can't agree. If even some groups that have found XMRV and say it's a real virus that's in the population (like Switzer's and Singh's) say it's not in the blood or they can't find it there, then i don't think one should use a different standard for ME/CFS. Why look in a way that seems to be the least good? Ok, it is true that the positive ME/CFS studies have reported finding it in the blood and if those were wrong then why should it be in ME/CFS subjects at all? Maybe because it's a type of virus that would fit. And even if there were no reasons i would look.

    I find it rather unlikely that Silverman will distance himself from his prostate cancer findings, but we will see. Maybe you have more information than me. What i don't see is how this would be a smoking gun in relation to the WPI's findings. Haven't they said that there never was any 22RV1 in the state of Nevada?

    What do you mean with "genetically distinct" virus?

    Unfortunately, it does not seem to be only science that is influencing the course of things, in my opinion, so we have to be careful and keep our eyes open. And even more importantly, develop better capabilities to influence things ourselves.

    As far as who would be to blame if it was to turn out that the WPI was wrong, i personally would not let them get away as easily. But this is not what has happened and not what i expect to happen, so that's strictly hypothetical. In fact it's the 20th and so i will even donate to them today ;).

    I agree that XMRV will not be something that will be forgotten quickly, no matter how things go on from here.
  15. jstefl

    jstefl Senior Member

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    This whole XMRV in the blood confuses me greatly.

    There seems to be at least some agreement that XMRV exists and can be found in the prostate or other tissues. My question is how did it get there if not thru the blood?

    I don't see how the virus could enter directly into the prostate from outside the body, so is there any other path it could have taken?

    It seems to me that Dr. Singh, and many others need to answer this question.

    John
  16. eric_s

    eric_s Senior Member

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    Yes, agree. Maybe it's not always present in the blood or only at very low levels or in a form that's hard to detect.
  17. Gemini

    Gemini Senior Member

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    Excellent point. Has anyone requested, in writing, CDC do ME/CFS tissue work? If so, perhaps they could post the CDC's response? Be interesting to see what their position is.
  18. LJS

    LJS Insert Witty Comment Here

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    I do not think this is a valid argument, the original science paper found it in blood and the WPI continues to say they can find it in blood. Heck look how many people come back positive through VIPdx with a blood test, we are not sending tissues off to them. This is getting a little ridicules at this point. If others can not find XMRV in blood it does not mean that it is hiding in tissue; it means the WPI is wrong and their lab is contaminated. If you look at the sequences the WPI posted some are 99.9% identical to 22Rv1 and 99.4% identical to VP62, the WPI are not finding something radically different then what all the tests are designed to find. It would be easy to argue from the sequences the WPI published that they are in fact picking up a lab contaminate due to the fact they are almost identical to the lab strains of XMRV. The Singh lab should have been able to easily find XMRV, if it was there, given the WPI sequences.
  19. eric_s

    eric_s Senior Member

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    No, it just means that after a certain point others can't find it in blood. Maybe contamination is the more likely explanation (i don't share this view though), but it might also be that the others just are not good enough at looking.

    And don't forget some others have found it too. Lo, Hansen and IrsiCaixa. Also RedLabs is finding it and even though they have a license, they are an independent lab from VIP Dx. Hopefully now Dr. Bieger has also found the virus, we should hear about the results any minute now, i guess.

    Probably there's much more of it somewhere in tissues and so this might help the others finding it, as it's easier there.
  20. Esther12

    Esther12 Senior Member

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    I guess it's possible that the WPI's science paper on finding it consistently in the blood was wrong, and the result of contamination... but that XMRV is coincidentally related to CFS and needs to be looked for in tissue samples. The would be funny, but is a bit of a long shot.

    With that exception, I agree that it's the ability to find the virus in the blood of CFS patients that made us think there's an association, and without that, there's no reason to think that there is an association. It would be good to hear news from the BWG soon. Their last set of results didn't really do much to clear matters up for us.

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