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XMRV results

Discussion in 'XMRV Testing, Treatment and Transmission' started by Patient 2, Dec 1, 2009.

  1. kurt

    kurt Senior Member

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    Good memory Kim. I maybe talk too much sometimes. Yes, I mentioned that I have been told the preliminary results of two replication studies. And I have heard about additional studies also. Sorry I can not say more than that right now (literally, that was a condition of my being part of a study).

    As for VIPdx, who knows. Anyone can be mistaken with a new test that has not been validated. VIPdx or CD, or WPI, or the Berlin study, ANY of them could be producing false results. So do I think it is possible VIPdx is getting false positives? Absolutely it is possible. But is that happening? I don't think anyone knows at this point.

    Here is what I understand. WPI used a standard PCR. A standard PCR is subject to false positives. WPI also used the MuLV antibody for their antibody and culture studies, and NOT an XMRV antibody. So they are relying on a cross-reaction of MuLV, which gives some risk of false positives. However, WPI sequenced two antigens for XMRV, that is hard to explain away except by contamination of media or sample.

    CD and, I assume, the Berlin study and others are using a real-time PCR test, which has very low risk of false positive, but some risk of false negative.

    So any of these studies could be wrong at this point. That is why this will take time and many replications are required.

    I agree, regardless of the final outcome for XMRV, we DO have some evidence of retroviral problems, the May presentation of WPI Virachip results showed that, both HERV and HTLV. And also multiple HHV infections, far more than controls, and that is really interesting as HHV can activate HERV from our DNA. So it is time for the world to pay attention to the reality that CFS is a serious co-infection illness with immune suppression. For me that is the BIG take-away from all this.

    Very good questions. Wish I could answer them... My understanding is that replication studies need to test both the same sequences and different sequences from the original reports. If the virus is there, all sequences tested should be found.

    Well you are entitled to your opinion. I know enough about Cooperative to respect their science, so until someone can prove them wrong I will believe them just as much as WPI or the Berlin study or the CDC study (the one by the retroviral research group) or any other competent lab.

    Cooperative is in the business of rapid test design, so I think they deserve the benefit of the doubt until their report is published and reviewed.

    I can not say anything about my personal results right now due to an NDA.

    **

    If XMRV is really there in 98% of PWC but WPI has not revealed enough about their testing for others to easily replicate their work, then they will have a problem to deal with. If XMRV is not present in PWC then WPI will have to explain what happened in their studies and move forward. The world will not end, this is ordinary science. And hopefully in either case the truth will emerge from the debate and ongoing replication attempts by the scientific community.

    Clearly we all need a rest from the suspense and I am doing all I can through my contacts to encourage rapid publication of one study. Hopefully some of the other completed studies will report soon as well.
  2. cfs since 1998

    cfs since 1998 *****

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    WPI PCR found 68 of 101 patients positive and 8 of 218 healthy controls. How can this be explained by lab mistakes and false positives? The probability of that is almost zero. Science wouldn't have published it if false results were as likely as you say they are.

    Apparently you think that the chance of WPI being wrong and the chance of Cooperative Diagnostics being wrong are equal. Which makes no sense, logically speaking. WPI's test found an enormously significant difference between CFS patients and healthy controls, CD did not. WPI's results were published in Science, CD's have not been. CD has offered zero evidence in support of their test. CD has not established that their test can distinguish between CFS and healthy controls. CD tested 400 samples and did not get a single positive. I would say they have no credibility but since they offer no evidence, there is nothing to judge as credible or not. For all I know a Magic 8-ball would be as accurate as CD's test.
  3. fresh_eyes

    fresh_eyes happy to be here

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    :D

    And let's not forget that WPI didn't do all the testing in the Science study - Cleveland Clinic and NCI would have to be mistaken as well. VERY, very, very unlikely.
  4. _Kim_

    _Kim_ Guest

    Not necessarily. The CC and NCI reproduced the results using the same testing procedures (standard PCR) that WPI used.
  5. Smulan

    Smulan

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    But if they also tested healthy controls and got the same ratio its hard to belive a false postiv answer theory.
  6. fresh_eyes

    fresh_eyes happy to be here

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    But by that reasoning any successful replication studies will be questionable too, since a good-faith first replication effort (like we're hoping for) means they'll use the same methods and the same samples. Where does the doubting end?
  7. GoWest

    GoWest

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    standard PCR questions

    Kurt, if I am understanding a standard PCR, the risk is that a small amount of contamination will lead to a false positive. Is this the concern you have for the standard PCR? Given the extreme difference in results between healthy cases and cfs cases I think contamination is most unlikely. If you get a positive on a PCR without contamination evidence you can bet the farm the patient is infected.

    One of the problems we used to worry about with the mycoplasma incognitus testing was that the bacteria would disappear if the blood was not sent on dry ice and tested asap. I don't know if this would be a concern with a retrovirus.

    Paula Carnes

    This is a quote on the problem with standard PCR:

    Preventing carry-over contamination with uracil-DNA glycosylase (Roche Diagnostics Corporation)
    PCR can amplify a single molecule over a billionfold. Thus, even minuscule amounts of a contaminant can be amplified and lead to a false positive result. Such contaminants are often products from previous PCR amplifications (carry-over contamination). One common strategy to avoid such contamination is substituting dUTP for dTTP during PCR amplification, to produce uracil-containing DNA (U-DNA).
    http://www.roche-applied-science.com/pcr/applicati...
  8. _Kim_

    _Kim_ Guest

    WPI used more than 3 MuLV antibodies. Is it really possible that they got false positives from all three?

  9. Cort

    Cort Phoenix Rising Founder

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    Theres always the question of statistics. Maybe I shouldn't have said anything because we wont REALLY know about XMRV until a series of research groups do statistically valid studies. Lets say the doctors patient base really had a 60% positive rate; even then at some point he's going to get a long run negatives or a long run of positives; statistically thats a certainty.

    We also don't know how that doctor diagnosed ME/CFS patients. Dr. Komaroff reported a study he did at the last conference where, if I remember correctly, that 50% of the patients referred to him as by other doctors as having CFS didn't even fit the standard definition of the disease. He was able to show that the CFS patients did have abnormal EEG results, I think it was- the fatigued but non-CFS patients did not. The same could very well apply to XMRV.

    This is why we need multiple rigorous studies with well defined groups of CFS patients and validated tests before we really know whats going. (Its also why I should probably keep my mouth shut about these emails -( its hard though :))
  10. Advocate

    Advocate Senior Member

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    Hi GoWest,

    I see this was your first post. It's good to have you here!

    Advocate
  11. kurt

    kurt Senior Member

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    There are multiple possible reasons for a false positive result, I do not know them all. Testing artifacts are possible, for example. That means something in the test is wrong, in the reagents perhaps, or the way they combine with the DNA of the patient's own cells. Some combination of multiple problems can probably produce false positive results that look like a real result, including differences between test patients and controls.

    Also, there is the 'wrong bug' type of false positive. That actually seems most probable to me. In other words, some other bug has part of the XMRV sequence. That could present exactly as their results showed, but not actually be XMRV.

    Also the tested cohorts could be different. WPI used some biomarkers to select their test subjects, CD and other replication studies will probably just use a sample of ME/CFS patients.

    If a lab is pursuing an agenda, trying to build a case for something, they will sometimes see what they are looking for, basically cherry-pick their procedures or selection criteria until they get the results they want. Sometimes they inadvertently discover a new way to create false positives. This is a known risk in all forms of research, I was a researcher myself before CFS and have had to address this issue. The only way to determine whether initial findings are accurate is through replication attempts, particularly by outside researchers using their own (different) methods. Not matter how strong an initial finding looks, it can fall apart. I like to mention the 'Cold Fusion' debacle as an example, that research looked very solid, that is, until the replication studies failed. And at one point one of the Cold Fusion research teams did get published in Science... So Science has made mistakes before, they are an interdisciplinary journal. They are not experts in everything. Also, they can not be expected to figure out something that only a replication study will reveal.

    This issue will be settled through the scientific process, and not by our reasoning or beliefs, including my own. What will happen may not be what people who are advocating for or against XMRV as the cause of CFS might like to hear.

    EXACTLY, that is the way this works. People will also question the replication studies, as they should. The doubting will not end until a consensus finding begins to emerge from many different studies.

    The reason I am posting at all is related to what you say, people don't like the 'doubting.' As if doubting XMRV is somehow doubting CFS. Nothing could be farther from reality. We must doubt any bold claims about CFS until they are proven, and one article in Science is not proof. Dr Coffin made that clear at CFSAC, he knows the WPI study was good, but still just the opening play in a very long game. He has been there and done that before...

    I addressed that above, contamination is not the only possible cause of false positives. If there are conflicts between studies, this will have to be figured out.

    Absolutely yes, because they used MuLV antibodies and not XMRV antibodies. MuLV antibodies are a type of general test for XMRV, evidence of some related infection. So WPI is obviously counting on cross-reactivity between MuLV antibodies and XMRV. By definition this type of approach to antibody (And culture) studies can produce false positives. What that would mean is that some other antigen (not XMRV) is present that reacts with MuLV antibodies. That is very possible, in fact there are even endogenous human retroviruses that are similar to MuLV, that are proven to be present in CFS (some HERV classes are considered gamma retroviral). WPI showed that in their May presentation. There could even be some novel unknown antigen causing false positives on the MuLV antibody test. This is the point that has mystified me the most about the Science article. Why they did not address the risk of their antibody test for cross-reactivity thoroughly, that is a big issue. Maybe they did address that off-line and just did not report it. I think we will learn more about WPI's actual testing process when the first replication studies appear and WPI defends their study.

    Final thought about testing - until multiple replication studies are reported and some type of consensus emerges about XMRV, any testing is really for research purposes only. I think some people are counting on a positive XMRV finding to help validate their ME/CFS, and to point to a new treatment. I hope they realize they are gambling at this time. Personally I would wait for a validated test before investing my hopes (and money) on XMRV testing. That may take a year, given that some replication studies will not report until Spring and some are just getting started so probably will not report until Summer or Fall of next year.

    Addition: Just found an interesting study showing that MLV antibodies are considered cross-reactive with HERV. In this study the researchers studying psoriasis used MLV to illustrate that HERVs were active. So clearly MLV is known to be very cross-reactive... see: http://www.ncbi.nlm.nih.gov/pubmed/12902038
  12. fresh_eyes

    fresh_eyes happy to be here

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    I get what you're saying, Kurt. I really appreciate you making the effort to explain where you're coming from, and it's definitely good for the forum to have a variety of perspectives.
  13. Cort

    Cort Phoenix Rising Founder

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    Correction! I made a mistake with that 11 negative results post; the doctor used VIP dx to test his patients - not Cooperative Diagnostics. My apologies.
  14. lebowski

    lebowski

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    " Originally Posted by fresh_eyes
    But by that reasoning any successful replication studies will be questionable too, since a good-faith first replication effort (like we're hoping for) means they'll use the same methods and the same samples. Where does the doubting end? "

    the doubting will not end even when they can treat cfs with some haart like therapy as they treat aids .. the doubting is still pushed by some " feeding with negativism " type of people for aids , u can see it if u google for " it s not hiv " ..
    let me put it this way , after ten supporting reasearch , there will be people still tring to show the whole thing as there is fifty percent possibility of xmrv really involved with cfs , or there is a real retrovirus in pwc ..
  15. Jimk

    Jimk

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    Cross validation

    I know of one highly credible medical center lab that tried to replicate the PCR test based on the published protein sequences in the Science study. This is something that should be easily possible for a professional lab to do and is the reason why the long lists of protein sequences are published in such studies, so that the work is replicable to anyone with the skills and equipment.

    Four patient samples, all diagnosed with CFS, came up negative. The lab head told me that they considered this unlikely given the WPI/Science results, but without a validated XMRV sample to "test the test" they can't really know if they erred in the test they developed or the patient sample was truly negative. They've sent to Cleveland Clinic to get a sample of XMRV DNA (they don't send actual virus anymore evidently too risky and not needed) so they can perform their test on the actual substance they are testing for, and this could take a month or two to go through procedures.

    All this to underline the point that even a "regular PCR test" is not always a simple matter, and unless we know that a test a lab is doing has been properly validated we don't really know what it's measuring. The same holds true in any replication studies of course.
  16. fresh_eyes

    fresh_eyes happy to be here

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    Well, perhaps Reeves and Wessely can get a position under Thabo Mbeki...:)
  17. lebowski

    lebowski

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    " Well, perhaps Reeves and Wessely can get a position under Thabo Mbeki..."

    :eek:

    i promise myself i will not die till the day i see all the psyhciatrists r out of bussiness who r treating infected people with woodoo talking and lifetime symptom relievers .. ( and gradual immunesuppresing exercise and light streching and hard streching )
  18. Marylib

    Marylib Senior Member

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    for Sue/Ladybugmandy

    I think it is Sue -- my memory is shot.

    I wish I could say something that would help you somehow! I know how fragile you must feel now. In a few months we will surely know more about XMRV. And I believe even if XMRV is not the only culprit, we are finally coming out of the darkness we have been in so long. I think there will be help soon. And I hope that people like you, who are just hanging on, will get that help very very soon.
  19. fresh_eyes

    fresh_eyes happy to be here

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    I know, Sue. I know, I know, I know.

    Perhaps antibody test results, when they're available, will look different.

    Hang on, Sue. We're all thinking of you.

    (Perhaps we can all learn from Lebowski, whose desire for revenge on Wessely et al seems to be keeping him going! :D)
  20. Martlet

    Martlet Senior Member

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    I can only imagine how you are feeling.

    I am trying not to pin too many hopes on XMRV research (although I did when the news first came out) but see it as possibly another piece in the puzzle, certain that one day we will have the entire picture.

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