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XMRV Study No. 4

Discussion in 'XMRV Research and Replication Studies' started by Orla, Feb 25, 2010.

  1. Advocate

    Advocate Senior Member

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    CFSACMeeting CFSAC Day 1 Annette Whittemore

    Hi Gerwyn,

    I wouldn't do this for just anyone, but for you I have transcribed Annette Whittemore's testimony at the Chronic Fatigue Syndrome Advisory Committee's meeting last fall. It was expertly taped, thanks to Wanda Jones, and is now on YouTube.

    I transcribed only as far as the statement you are looking for, but if you need the rest, I will transcribe the rest. (I suspect that you are a hunt-and-peck typist, and I am a speedy touch typist.) Maybe someone has already transcribed it, and I just couldn't find it.

    I see that when I quoted her above, I did not get the words exactly right.

    http://www.youtube.com/watch?v=oxF-PV3z0Do&feature=PlayList&p=CFEE74155E8172E6&index=11

    I see that Annette Whittemore's statement is almost identical to what jspotila wrote, so maybe I'll add her to my refrigerator door gallery also. I look forward to visitors' questions: "Who are all these women?"

     
  2. jspotila

    jspotila Senior Member

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    :eek: Advocate, you want to put my picture on your fridge? I have to admit, this is a first!!!! :D
     
  3. Advocate

    Advocate Senior Member

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    I wonder if it's a first for Wanda Jones and Ila Singh. I'm getting quite a collection.
     
  4. kurt

    kurt Senior Member

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    The point of a validation study is simply to find the virus, they do not have to follow the exact protocol, just a protocol that they believe is sufficient to find the virus. And that protocol worked with positive controls so they could find that virus. That 22Rv1 study you mentioned showed that there are many copies of the XMRV virus in an infected cell, both in RNA and in the chromosomal DNA, right? So if an XMRV infected cell was present in the Dutch study there would have been plenty of matching sequences and XMRV should have been found since they used TOTAL nucleic acid extraction, which is both RNA and DNA. I realze the Dutch study talked more about RNA, that was because that required special processing, the DNA was still extracted though and tested. In fact if you read my earlier quote from a retrovirology testing expert, it was a very strong finding, the Dutch study went through a better process than the two UK studies. Was it perfect, no, I agree with that, they did not fully confirm RNA viability, and if the Dutch study had only tested the RNA, then the entire experiment would be invalidated, as you have stated. But the DNA was also tested and the negative finding of XMRV well validated. They even tested cDNA, in an extra step, along with the DNA, so it was amplified.

    Could there be cohort problems? This was a well tested and profiled CFS cohort, used in other studies, in the early years of CFS, so I think it might have been a good cohort. But that is one of the possible criticisms of this study, the cohort may have not been the same. Until WPI releases more information about their cohort that is impossible to compare.

    I agree that a blinded study is a good idea, wonder if WPI would agree to that, probably would require two labs. That was done by the CDC with DeFreitas, maybe we will end up in that same place eventually with XMRV.

    I have another response from my expert contact and will post that soon, I am trying to use non-technical language, but when it comes to the biological explanation he can explain this far better than I can.
     
  5. kurt

    kurt Senior Member

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    For Gerwyn and others interested in explanation of Dutch study RNA problem

    Gerwyn, Here is more feedback from my contact as mentioned before, to respond to some of your comments about the 22Rv1 study and RNA problems with the Dutch study.

    "You have read the paper "Multiple Integrated Copies and High-Level Production of the Human Retrovirus XMRV (Xenotropic Murine Leukemia Virus-Related Virus) from 22Rv1 Prostate Carcinoma Cells" by Knouf et al. In it they observed viral budding underneath an electron microscope. What causes the viral budding? Viruses breaking out of the cell. Where do these newly born viruses come from? They are created from the DNA integrated into the cell's genome. In fact, the very title reads: "Multiple Integrated Copies" referring to the number of copies of XMRV present in every single 22rv1 cell. There are roughly 6 pg of DNA in a single 22rv1 cell. If Knouf et al are correct, there are at least 10 copies of XMRV DNA in those 6 pg. Therefore, PCR using 250 ng of 22rv1 DNA would have 416,666 copies of XMRV DNA present. Since PCR is capable of detecting as little as 1 copy of viral DNA, it is more than capable of detecting 416,666 copies! In fact, PCR has always been more sensitive than detecting viruses under an electron microscope. Although you can see a single virus budding under the microscope, there have to be millions present to observe just one. It's kind of like finding a needle in a haystack - if the haystack is full of needles, it's very easy to find one! If not, then you may have to resort to a more sensitive technique like PCR to find it, figuratively speaking."

    "As to DNA versus RNA detection, you are right about the RNA detection. They used the phocine distemper virus as a control to ensure that the sample preparation steps did not destroy the RNA/DNA present in the sample. However, they did not prove that any RNA was in the samples before the extraction. They did, however, prove that the cellular DNA had been preserved in the samples and was still present after the "total nucleic acid" extraction (total nucleic acid extraction isolates both the RNA and the DNA). They did this through amplification of the beta-globin gene. Performing a reverse transcription step to create copy DNA out of the RNA does not destroy or even alter the cellular DNA present. In fact, the purpose of a "total nucleic acid" extraction is to be able to detect both RNA and DNA from the cells. The PCR that is performed on the mixture containing copy DNA also amplifies the cellular DNA. The only problem with this method is that it does not allow differentiation between what was previously an XMRV RNA virus and what was viral XMRV DNA integrated into the cell's genome. However, since there were no positives, Kuppeveld, et al did not have to make that distinction. Not only was there no XMRV RNA present, there was no XMRV DNA present. Since the cellular DNA was fully preserved, the absence of XMRV in the cellular DNA means that the cells in this study
    were not infected with XMRV."
     
  6. Gerwyn

    Gerwyn Guest

     
  7. ukxmrv

    ukxmrv Senior Member

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    Kurt, Gerwyn and all,

    A comment from the Plos One paper (McClure, Kerr, Wessely one) from Dusty Millar on the beta-globin gene
    ==================================================================

    In response to your first paragraph, it wasn't clear from your statement in the Results section of the paper that detection of single copy XMRV DNA was performed in the presence of patient DNA, only that a single copy could be detected in "the reaction". And again, your ability to amplify the relatively abundant beta-globin DNA is not the best way to show that you can amplify DNA present at much lower amounts.
     
  8. Gerwyn

    Gerwyn Guest

    exactly you could amplify something but GOK what
     
  9. Gerwyn

    Gerwyn Guest

    thankyou very much I really appreciate it
     
  10. kurt

    kurt Senior Member

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    Gerwyn and others, I just do not have time to continue this discussion. So please do not interpret my lack of response as agreement with the points being raised here. I do not agree with this, and in general I am finding a pattern here that some of your points are valid and some are not, but just do not have time to sort through it all for every comment made. I wish I did have that time because you have found a few issues that others have missed.

    As for this statement, I disagree right from your first comment. You do not need replicating virus to find DNA by PCR. PCR is far more sensitive than culturing which requires thousands of viral copies just to get one to grow, a PCR test can find one dead virus, PCR can amplify the DNA in dried blood (forensics), cave man bones or even dinosaur fossils hundreds of millions of years old !! And that is just a bit older than even the Dutch study samples...

    I don't have time to comment on the rest but the whole argument just does not work. Some of the points you mention make sense but according to my information it does not add up the way you say, and I just don't have time to research the points to explain this well enough.

    You have asked before why I do not look at the flaws in the replication attempts. Fine, the flaws in the Dutch study are primarily the lack of verifying the extracted RNA was viable and a possibly biased sample cohort. Regarding the RNA, there is no proof the RNA was not viable, they simply did not prove that it WAS viable, it might have been fine, there was some evidence of that, they were satisfied it was alright. And I don't think we can fault the cohort much, as so much less was known about CFS then, they did have some very sick people and I think we have to let that one go. There were enough samples that if PCR shows XMRV in 67% of CFS patients and XMRV is necessary for CFS, there should have been many positives. So I agree with those two flaws to a limited extent, but do not agree the DNA was flawed, there was nothing reported there that would invalidate the study, I think we have to accept that the results do not validate the WPI Science paper. That is the only accurate conclusion that can be drawn. That is a good way to state the facts without any anti-ME/CFS 'Spin'. Definitely they did not prove that there is no XMRV in Holland TODAY. But if CFS has not changed in many years we have to assume that the study weakens the WPI finding. Still, a consensus process is required, and this is not enough information, I would agree that more studies will have to shed light on all the subtleties of XMRV in CFS before definitive statements can be made EITHER WAY.

    To say XMRV causes CFS is just false at this early point, that is not a consensus view, and not a good basis for advocacy of public policy changes right now, and patients should not be getting their hopes up yet. In my opinion encouraging hope of a cure from an early finding like this would be unethical when there is no verification from an outside lab (which REQUIRES running different test designs), no validated causal models, no treatments to test, no clinical trials, and certainly no successful stories of remission or cure yet.

    To say that XMRV is disproven as some in the UK have done is also false at this point, again, a consensus view will be required. I am trying to write a little about a consensus process model for that right now...what will be required for consensus (operative word is 'trying', this is a difficult and amorphous topic in science).

    Certainly I am just N=1 as you have pointed out, and so are my sources of information here, but so are you and so is every researcher here or elsewhere with an opinion about XMRV. Thus the reason for a consensus process, and the reason I try continually to encourage forum members to maintain an open mind about XMRV. The future remains unpredictable although clues are starting to come in.

    The total nucleic acid extraction process by definition includes the patient DNA, so it had to be present. Lower quantities would not deter PCR detection, they should have had more than ample copies if XMRV is present in CFS patient blood at the concentrations found in the WPI article. Nearly half a million viral copies if you do the math, and only one is needed for the best PCR tests. PCR is the gold standard in testing, not culture or serology (antibody) studies that are more prone to cross-reactions and false positives and are non-specific.
     
  11. ukxmrv

    ukxmrv Senior Member

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    Kurt,

    You really have been very patient with us and I appreciate that. You have spoken to people and found extra information, then been kind enough to try and help those of us struggling with this, to try and improve our understanding of the arguments. I would not like you to think that I wasn't grateful.

    Thank you!!
     
  12. V99

    V99 *****

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    If the Oxford criteria shows CFS to be in 2.6% of the population, and fukuda about 0.24 to 0.42% of the population. This is a 6 to 10x increase roughly. 32 patients were in the Netherlands study. Therefore between 3 to 6 of these should have met the Fukuda criteria. Is this not a low number? And what if you then take into account the Canadian criteria?

    I'm no expert, but are my figures correct?
     
  13. Gerwyn

    Gerwyn Guest

    Kurt do the science this is not opinion RNA degrades frozen 15 months best so far T and B cells best so far 2 years neither 20 years.Cell dna may be present but no activated PMBC no replicating Ban d T cells no virus DNA.22rvi not cultured all virus integrated PCR useless

    the CBT enthusiast who diagnosed the patients did so according to criterea cobbled together in a lunch meeting pharma sponsored some two months earlier which expressly exclude anyone with the ccc symptom complex.He chose to ignore the Holmes criterea which were internationally agreed at the time in favour of unvaalidated criterea concocted by FOUR british pychiatrists with big axes to grind.Please dont say that the cohort was not an issue

    The KEY ISSUE IS THAT PCR NEEDS REPLICATING VIRUS TO WORK IT MAY BE GOLD STANDARD BUT NO PROVIRUS NO CIGAR.The conditions for replicating virus were not met in the experimental group or the controls
     
  14. Esther12

    Esther12 Senior Member

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    I'm pretty sure that no-one knows XMRV is the cause at this point. The WPI seem to think that they know, but lots of researchers have turned out to be wrong in the past, and their work is still at a really early stage.
     
  15. cfs since 1998

    cfs since 1998 *****

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    With all the evidence suggesting XMRV is so hard to detect, I'm a bit worried that we'll end up having the opposite problem and will end up finding XMRV in healthy people.
     
  16. Esther12

    Esther12 Senior Member

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    In my ignorant opinion, that's the only way you'd find it in 97% of CFS/ME cases - if it was really widespread, but due to genetic reasons and triggered by immune/whatever problems, some people had it become a more serious problem. I just don't see how a retrovirus could spread like CFS seems to unless there were an awful lot of carriers.

    We've still got a lot more waiting to do...
     
  17. kurt

    kurt Senior Member

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    There is no way to really know how many of the Dutch CFS samples were 'real' CFS but they all had serious clinical signs and symptoms per the study. Also, that is about an 8x increase if you take the average for fukuda. So IF the samples were in fact only meeting MINIMAL Oxford criteria, one would expect a minimum of 5 serious CFS cases , and at least 3 of those should have been positive by PCR. However, it is also likely that the samples might have all met a stricter definition, they only had Oxford at that time to talk about, so to assume that there were marginal patients means you are speculating that the study included some mild symptomatic patients, which does not seem likely given this was a cohort selected for study.

    No, that is not the key issue and not even correct as I understand retroviruses. Maybe you are thinking of a DNA virus but RNA viruses (retroviruses) can be found in non-replicating form already embedded in host cell chromosomal DNA.

    You seem to be assuming that the people running these tests are incompetent and have ulterior motives and are intentionally putting together tests that will fail. That just seems outlandish for published research as publication puts one's reputation on the line, it just would be crazy for a lab to do that, they would look foolish when their finding is contradicted. I think more probable is that they read the Science study and realized they knew a quick PCR method that they could run inexpensively on samples they had available that should be able to isolate a retrovirus like what WPI described. They stated that they fully expected to be confirming the WPI results. The finding was a surprise, I believe that because I know that has been happening at other labs.

    How can you be sure of anything at this point about XMRV? There is no scientific consensus yet and this is very tentative early work. WPI has already proven that their original study can be replicated, through NCI and CC, so it is absolutely no surprise that they will be able to replicate their study again. But replication only proves that the method used works when tried again. More important is validation, which is to find the virus with a different test design and this is CRITICAL in this type of research as the standard PCR test methods used by WPI are known to sometimes produce false positive results. So if only one test design works and all others fail, that original design is suspect. And this is not rocket science, if the virus is there it should be found by many different test designs, there should be multiple PCR test designs that should be finding the virus at this point. I will be totally unimpressed if WPI simply runs their study again at another lab with the same reagents and has the same results. Someone besides WPI using their own reagents and samples must validate XMRV. If that is not possible, then that casts a shadow of doubt on the original testing. As for the outside labs not cooperating, I don't know about that but I do know that WPI has not answered a lot of questions others have had.

    This is not a fight, there IS NO FIGHT about XMRV. This is science and it will be decided using the scientific method and eventually the scientific consensus process. Like it or not there is absolutely nothing any of us can do to change or influence that. WPI does not need support in this, they are asking for financial support, that's business, but their science must stand on its own legs.

    And for the record I do not buy into the idea that outside researchers are somehow sabotaging their work and willingly ruining their reputations, careers and futures by publishing shoddy tests, researchers are simply too smart and not invested enough in ME/CFS to sacrifice their careers that way. They are taking at face value what WPI said in the Science article about how they found XMRV, trying a test that they believe should also find the virus, and discovering it does not work, they do not find the virus. They find the virus in their control samples, they find it in real prostate cancer XMRV samples, their tests work, but they do not find the virus in CFS samples. And some are running ultra sensitive tests, they should find XMRV if it is there.
     
  18. IamME

    IamME Too sick for an identity

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    Such as what? The study says:

    Patient cohort
    All patients and controls examined in this study were part of a Dutch cohort of 298 patients, which has been described in detail.10 11 All patients of this cohort fulfilled the Oxford criteria and reported severe, unexplained, debilitating fatigue of at least one year in duration.12

    These references are:
    # Vercoulen JH, Swanink CM, Fennis JF, Galama JM, van der Meer JW, Bleijenberg G. Dimensional assessment of chronic fatigue syndrome. J Psychosom Res 1994;38:383-92.[CrossRef][Web of Science][Medline]
    # Swanink CM, Vercoulen JH, Galama JM, Roos MT, Meyaard L, van der Ven-Jongekrijg J, et al. Lymphocyte subsets, apoptosis, and cytokines in patients with chronic fatigue syndrome. J Infect Dis 1996;173:460-3.[Web of Science][Medline]

    The first was a study into

    psychological well-being, functional impairment in daily life, sleep disturbances, avoidance of physical activity, neuropsychological impairment, causal attributions related to the complaints, social functioning, self-efficacy expectations, and subjective experience of the personal situation.

    The second they used to dismiss immune abnormalities:

    Endotoxin-stimulated ex vivo production of tumor necrosis factor-alpha and IL-beta was significantly lower in CFS. The immunologic test results did not correlate with fatigue severity or psychologic well-being was measured by Checklist Individual Strength, Beck Depression Inventory, and Sickness Impact Profile. Thus, these immunologic tests cannot be used as diagnostic tools in individual CFS patients.

    And the only "symptom" measures (typical for the CBT school) were fatigue and psychological.

    Is that what you mean by well studied and "serious clinical signs and symptoms"?

    And as for the Oxford criteria...

    http://www.myalgic-encephalomyelitis.com/oxford_criteria.html

    A syndrome characterized by fatigue as the principal symptom. ... Other symptoms may be present

    Exclusions contain:

    proven organic brain disease. Other psychiatric disorders (including depressive illness, anxiety disorders, and hyperventilation syndrome) are not necessarily reasons for exclusion.

    Post infectious and non-post infectious cases are lumped together, yet ME is post infectious.

    Fatigue and even disability are described as "subjective".

    The authors' views on disability are telling:

    Disability (eg inability to walk) should be distinguished from impairment of function (eg weak legs), and from handicap (eg unable to work)

    So unless you're paralysed, you can't be disabled according to Sharpe et al.

    The only symptoms mentioned in any detail (in the glossary) are fatigue, mood, myalgia, sleep.

    Sharpe's nname is prominent on the Oxford definition -- he & co would go on to remove all clinical signs from the 1994 Fukuda definition. Some of his claims include:

    Extensive physical investigation is unlikely to be fruitful and should be limited

    These unhelpful or 'dysfunctional' cognitions include the beliefs that recovery from the illness is not under personal control or that the illness is poorly understood.

    the use of extensive laboratory investigation may be psychologically harmful to the patient by reinforcing their beliefs about serious physical disease.

    Even if shown to be beneficial, such [immunological] treatment is unlikely to be feasible on a wide scale because of cost

    Excessive investigation should be avoided. Problems may arise if the patient requires a diagnosis the doctor feels is inappropriate or wants certification of permanent invalidity [i.e.] "ME"

    [Patients] higher levels of depression serve to reinforce the now widely current notion that such patients may be suffering from a depressive illness, of which physical fatigue is a somatic manifestation.

    The exclusion of persons [with psychiatric disorders] would substantially hinder efforts to clarify the role that psychiatric disorders have in fatiguing illness. We dropped all physical signs from our inclusion criteria [as] their presence had been unreliably documented in past studies. [Fukuda et al]

    Does CFS have biology? Yes not conventional disease pathology

    Clinically, it appears that interpersonal stress appears to be a major factor giving rise to development of CFS

    Those who cannot be fitted into a scheme of objective bodily illness yet refuse to be placed into and accept the stigma of mental illness remain the undeserving sick


    At the psychosocial Collaborative Conference (on CFS/ME: October 2007), Bleijenberg says "CFS is neither the result of an organic disease or ongoing exertion nor alleviated by rest.

    The people being studied by Nijmegen, Barts and KCL are not house/bedbound, and often willing to do CBT/GET so when they say "severe" or "disabled" that needs to be taken with a generous pinch of salt.

    So, as you can see, using the Oxford definition presents severe bias against finding a severe, diseased cohort and is ideologically motiivated.
     
  19. Gerwyn

    Gerwyn Guest

    Try finding Integrated retrovirus with PCR look it up! Endoretroviruses can indeed be "Found" but not by PCR again look it up!
     
  20. Angela Kennedy

    Angela Kennedy *****

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    Here here. Thank you very much for this IamME. This is also the sort of issues that have arisen with the Erlwein et al paper. 'disability' was mentioned using a scale used for mental disorders, for example, there being no actual physical impairment measured; the patient cohort appear to have undergone, as it happens, quite a lot of tests BUT ONLY TO RULE OUT PHYSICAL DISEASE - which seems to be in complete contrast to how clinical patients that the cohort was selected from are treated (a plosone responder shows that).

    The Groom study's cohort has very little information on the cohort- funny that the WPI is being criticised on that in the circumstances - and again, someone tells us we should accept there were likely 'Canadian' severely impaired types on faith - in this case Charles Shepherd... "There 'must 'have been some..." he said. Again, Fukuda - which proscribes investigations for clinical patients by the way, saying a mental health assessment is the acceptable minimum investigation, was used, at least on some of them. Mary Schweizer shows how easy 3-4 symptoms can bring in people not in a chronic infective state and without organic dysfunction.

    The 'chances' of finding a serious CFS case in these cohorts seem to be extremely low at best, and the numeric projections being bandied about are not even based on probablity theory, just speculation and faith. It is like asking, "How many penguins will you find in a cohort of ostriches?" "Surely, there's got to be some, right?"

    This is extremely flawed science, performed by scientists which include those with massive conflicts of interest, namely their own pet theories, of which money and academic status is heavily invested. Performing flawed science won't cause them problems (especially if they can use ad hominem and appeals to authority to silence critics such as sufferers or advocates, or even other scientists like Mikovits et al) but say, showing the cbt model/psychiatric paradigm/or even genetic/stress link is wrong (as a 'smoking gun' of a virus would) will cause terrible problems in terms of career. We cannot ignore that.
     

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