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Article: From the PCR Side: the Cooperative Diagnostics XMRV Interview with Dr. Brent Satterfield

Discussion in 'Phoenix Rising Articles' started by Phoenix Rising Team, Mar 17, 2011.

  1. eric_s

    eric_s Senior Member

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    Is this really true? I think the WPI has said that they have found a greater variability in the meantime. It has not been published yet, i think, but that does not mean they have not found it. Of course it's very important to publish...
     
  2. Cort

    Cort Phoenix Rising Founder

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    Right - should have said 'publish'....:cool::cool::cool:. In the Nature article Dr. Mikovits said we can show the variability...

    Definitely - gotta publish...it's the only way...
     
  3. insearchof

    insearchof Senior Member

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    Thank you for going to the trouble to compile this Cort.

    I agree with ixchelkali comments. The article though raises a lot more questions for me than it has in terms of providing answers. But that seems to be the status quo right now.
     
  4. jace

    jace Off the fence

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    Cort said
    Well now...
    I was talking about how they calibrated their PCR test, as you know. I do not intend to touch on other aspects that I have dealt with before here http://forums.aboutmecfs.org/showth...-study-is-this-new-looks-to-be-from-CDC/page6 (post 54 is fun ;) )

    Satterfield calibrated to the VP62 clone. It says so in the paper. Fact. But what Urisman did and Lombardi followed was to use a microarray technique. That developed a PCR assay which was able to detect XMRV provirus in a person which was known to be infected.

    Sorry? Alter and Lo's paper has been criticised on this forum for finding other members of the Human Gamma Retro Virus family that differed from the VP62 clone sufficiently that they didn't call them XMRV, but there was a 95% homology. Lombardi found VP62, VP35 and VP42, and since those papers were published Mikovits has stated that she's revisited the samples and found greater variability. So I really don't understand your quote, above.

    Remember, retroviruses can have as little as 85% homology, and still share the same name.
     
  5. Megan

    Megan Senior Member

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    Thanks Cort for the interview, the questions were thoughtful and covered most of the issues people have been raising. Like others have said I am waiting to hear a response from Mikovits, Ruscetti, Singh etc.

    One thing I dont want to lose sight of in this whole thing is that even if it turns out not to be XMRV, is there something else that the positive studies are picking up in CFS patients that is not in controls? Obviously this was the case with the lo and alter study, but might it also be true for the others? I really dont care if i have xmrv, abcd or some other virus, if they have found something that is cross reacting to our blood then the question remains what is that?

    Along these lines, this quote from Satterfield was interesting:
    For a bunch of people who are still battling the perception that this illness is not real, any ability to distinguish CFS blood from non CFS is still a huge finding, the above quote seems to gloss over this.

    I am also finding the arguments about why there are different results in patients and control to be very weak. Perhaps this has happened historically, but I dont think there were so many studies in the rabbit paper example. What is remarkable about the positive PC and CFS studies is how consistent they have been with each other. The PC ones are consistently finding between 20-30% in patients (yet another such study out today) and the CFS studies much higher rates. With respect to control samples, all together there are about 10 studies showing low infection rates in controls (<=10%). If this is all contamination then shouldn't these figures be all over the place? how could they all be getting contaminated consistently at the same rate? particularly if it is happening from so many different routes as is being suggested?
     
  6. Esther12

    Esther12 Senior Member

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    Certainly. I'm hoping the BWG and Lipkin studies will examine this thoroughly.
     
  7. Cort

    Cort Phoenix Rising Founder

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    Yes they can have as little as 85% homology but there isn't any evidence that it is true for XMRV and, on the contrary, there's a great deal of evidence that suggests it is not. For instance if you take samples of HIV from just two different people you've find scads of variability. In fact, if you take a sample of HIV from the same person at different points in time you'll lots of variability.

    However if you take XMRV from several people even if they live far apart from each other - which probably means the virus had to go through other people to wind up in both - the virus is virtually the same. That is true in prostate cancer patients and in the published reports of CFS patients. Yes, there are only two published, fully sequenced strains of XMRV from CFS patients but the Lombardi paper also stated that the env and gag sequences in all the positive patients were identical as well. (I imagine this refers to just a bit of the sequence, however).

    There is controversy about how to interpret Alter/Lo's findings - that's for sure. Alter/Lo and Dr. Mikovits believe they confirm the original findings; Stoye, Coffin, Raccaniello etc do not. One problem with the Alter/Lo sequences is that they are quite different from XMRV. If there is an XMRV swarm you would expect there to be a gradient of varieties - all of which kind of bunched together but the Alter/Lo findings suggest there's XMRV and then there's a big jump to the MLV's they found.

    That suggests they may not be related but you could look at it the other way - if Alter/Lo are correct then there is actually enormous variability in the XMRV group which still remains to be discovered.

    Dr. Mikovits report of increased variability, if it was significant enough, would change everything. It would either destroy the link between the 22RV1 virus and XMRV or it would suggest that that link stands but that XMRV had entered the human population. Proving XMRV is more variable than reported is vitally important.

    I wish they would publish their findings. I don't know why that would be a hard paper to get published. It just seems like a technical paper - you just follow this process and you get the result. The WPI could send the strains out to a well known lab - they do the work - and you've got a great paper.
     
  8. Cort

    Cort Phoenix Rising Founder

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    I asked Satterfield about this on the phone. My recollection is that he does believe that it is picking up evidence of infection. He does not believe that is picking up evidence of a different MLV infection but that it's probably picking another virus. It is a very interesting finding.

    Again there is the question, though, why other groups which are also looking for antibodies against MLV's have not found them yet in CFS patients. Could it be the cohort? Or is something else happening?
     
  9. Cort

    Cort Phoenix Rising Founder

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    I agree this is the weakest side of the contamination theory. After Dr. Mikovits said that it took them several tries to find XMRV in the original study you could say that extra handling made a difference but that's not true, so far as we know, in the prostate cancer studies. Its intriguing as well that VIP Dx reports lower percentages of positive patients at its lab which is what we could expect.

    You could also flip the 22RV1 finding on its head and say - if 22RV1 really is producing XMRV - and if 22RV1 is found in labs around the world then why isn't XMRV being found all over the world? Why are WPI and VIP Dx and the NCI lab the only ones able to find it - why isn't everybody picking it up? Theoretically if XMRV was a contaminant and has not infected people it should be readily found in both patient and control samples in equal amounts. The key that it was a false finding would be that it had very little variability.

    So, if XMRV is a contaminant then the question has to be asked why it showed in both the WPI and VIP Dx in such large amounts and not in other facilities?
     
  10. asleep

    asleep Senior Member

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    This is a common refrain from the denialist movement: there's something going on, just not this virus. It's a cheap trick to give the appearance of genuine interest while diverting the conversation and undermining the truth. It's the same technique used by politicians to sidestep questions. It's the same technique that parents use to avoid topics with their children. Now children, go along and play, papa Satterfield says there's nothing to see here.

    "Looking for antibodies" is a very broad, non-specific term. As usual, Cort, you gloss over all the details as if they don't matter. Cort would probably contend that "swinging at a baseball with a baseball bat" cannot produce a homerun since he once "swung at a baseball with a baseball bat" and struck out. Those pesky little details like "timing" and "making contact" would not matter to Cort.
     
  11. pine108kell

    pine108kell Senior Member

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    What you call a conspiracy theory, I call the scientific method. When one scientist claims to make an important discovey, others try to replicate it so that it can be validated and become part of scientific knowledge. Science doesn't work on faith or one person's word.

    If Dr. Mikovits had been as transparent as Satterfield was in this interview, we would have wasted far less time and money searching for a virus that is appearing less and less likely to have anything to do with CFS.
     
  12. Megan

    Megan Senior Member

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    Yes that is interesting. If XMRV does not work out then it is important that we dont let the above obsevations slip by.

    Since Satterfield has a personal interest in helping his CFS friends, is he going to follow up on what he thinks this infection might be?
     
  13. Cort

    Cort Phoenix Rising Founder

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    Actually that interview is full of details. If you'll check the antibody answer you'll see that Dr. Satterfield reported that the original study looked for an antibody believed to be present in both MLV's and XMRV. It sounded like the only option they had was to look for an MLV antibody. Since they antibodies have been created to the actual proteins found in XMRV. In your baseball terms the Lombardi swung a very broad bat that was designed to hit all sorts of things while the later papers swung a bat designed to hit XMRV. Actually the later papers, according to Dr. Satterfield, also looked for the same MLV antibody that the Lombardi paper looked for.
     
  14. lululowry

    lululowry Senior Member

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    Gerwyn helped me understand that there are at least two important ways that Satterfield et al. can be distinguished from Lombardi et al.

    First, the difference between PMBCs and plasma:

    Lombardi et al. (remember, this included three different labs- the WPI, the Cleveland Clinic and the National Cancer Institute) used western blot and IFC after 42 days of culturing peripheral blood mononuclear cells (PMBCs). They also used a range of antibodies, including SSFV, which is specific to MLVs.

    Thus, Lombardi et al. isolated DNA and RNA from concentrated PMBCs. For the detection of a human gamma retrovirus, the concentration of viral nucleic acid is vital.

    Satterfield, on the other hand, attempted to locate HMRV in DNA extracted from 62 microlitres of plasma (not PMBCs). Not only is plasma far more dilute than PMBCs (plasma is 97 percent water) but Satterfield used a mere 62 microliters. The concentration of potential HMRV viral nucleic acid in Satterfield’s assay would have been close to, if not actually, zero.

    At least Satterfield is consistent in his methods. Using 62 microliters of plasma for such a research study was reminiscent of his ridiculous commercial Cooperative Diagnostic test, which he claimed was able to detect HMRV in a finger prick's worth of blood before it was pulled off the market.

    Second, the Satterfield et al. assay:

    An assay that detects HMRV in Macaques monkeys will not necessarily detect HMRV in humans because antibody responses in primates are qualitatively and quantitatively different from those in humans.

    Satterfield’s assay was validated only for Macaques monkeys, not humans.

    In fact, Satterfield’s serology assay failed to find antibodies to HGRV in clinically validated positive human samples.

    So even if he had looked for HMRV in the right place and cultured PMBCs for 42 days, as Lombardi et al did, Satterfield’s assay was not validated to humans.

    Just three amino acid changes in the SU region would defeat the Elisa and Western Blot tests completely.
     
  15. FunkOdyssey

    FunkOdyssey Senior Member

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    Thanks for the explanation lulu, very enlightening.
     
  16. Cort

    Cort Phoenix Rising Founder

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    I don't think Gerwyn is correct. There is no indication in the Lombardi paper that they did any culturing for the PCR. I went around and around with him on this. I plucked out the methodology section piece by piece and showed it to him and asked where is the culturing? Finally he said it was inferred or something like that.

    If you'll read Satterfields interview a bit more closely you'll see he quantifies the amount of DNA he was looking through and according to him his tests looked through much more DNA than in the original study. Or you can actually look at the paper a quick look at which indicates the Satterfield et al didn't use plasma at all - they used the same PBMC's in the same manner as Lombardi et al.

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3050813/?tool=pubmed

    In the paper Satterfield et al point out how much DNA they examined

    They used the 22RV1 clone to test their samples against....

    The Lombardi paper stated the strains they found were essentially the same as VP62 and, I believe VP34 - which later research showed were essentially the same as 22RVI.

    There actually are no clinically validated positive human samples. That's the whole problem isn't it? Except for the labs in the Science paper no other labs have validated Lombardi's finding and that's how you validate tests. If there is a blood sample that everybody could agree was positive then they would all use that - but only the CDC and the WPI could detect XMRV in one of the BWG's tests and only sometimes (and the CDC not in the other one)....That's why Abbot spend $250,000 or whatever it is to infect those monkeys with XMRV. If they had had what they consider to be validated human samples they would have certainly used those to develop their antibody tests.

    The labs have, however, except for the culturing replicated all of Lombardi et al's methods. Neither the PCR nor the antibody tests involved culturing. I asked Racaniello about this and he said there's no culturing indicated in the PCR test in the paper.

    You know these people aren't stupid. This is what they do for a living and their labs credibility is at stake with every paper they put out. Gerwyn seems to rather consistently find glaring errors big enough to drive a truck through in his analyses of these studies; ie it was apparently impossible for Satterfield to find XMRV given their methods. But consider that given the attention this field is getting Satterfield and his lab are sure to look like idiots rather quickly if they make such silly mistakes as Gerwyn suggests.

    What Gerwyn is saying- that the negative studies are all bogus...and that the authors are idiots and they're making simple mistakes......is very enticing - he is saying what everyone wants to hear...But...

    If you read Satterfields bio at the top of the paper - his work creating new PCR techniques, his work with the Defense Dept using those techniques, his development of new algorhythms for PCR,... the fact that he founded a company that does nothing but PCR.....I feel like he probably knows what he's doing. I felt the same way with Dusty Miller and his 20 years of work in the field and his 200 plus papers. So if I'm going to have decide whether to trust someone; Gerwyn - or Satterfield or Dusty Miller - I'm going to have to go with them - no matter how enticing what Gerwyn says he believes is going on...

    That's just me! :) Maybe I respect authority too much! Time will tell.
     
  17. kurt

    kurt Senior Member

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    I don't think the above reflects an accurate understanding of either the science or the actual studies being compared. The WPI culture study was separate from and dependent on the findings of the PCR study. Gerwyn's comment as quoted also contains a serious factual error, they did not base their test on 62ul of plasma. Did you even read the article at the start of this thread? Here is a quote from the article that answers most of the points being made about the sample volume.

    now for the second part of Gerwyn's comment...

    Well, that is all we have that can be validated, the Macaques... to utilize a WPI sample for validation would be circular reasoning and improper in science. You can't use a sample for validation that comes from the test you are trying to validate !!! The sample used has to have external validation, and only the monkey study has that right now. Again, from the paper above:

    And the rest of the comment:

    Again, this is an incorrect statement. There are no clinically validated positive human samples. THAT IS THE WHOLE POINT of running confirmation studies, to validate the finding. This takes consensus, many researchers studying the issue with many tests from many angles. Science is not a one-man or one-lab operation anymore.
     
  18. ixchelkali

    ixchelkali Senior Member

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    Good point.
     
  19. ixchelkali

    ixchelkali Senior Member

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    Does anyone know how much variability is found in feline leukemia virus? Since it's another gamma retrovirus, it seems like it would be more analogous to XMRV than HIV is.
     
  20. asleep

    asleep Senior Member

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    This:

    Then this:

    Then this:

    Guys, if you're going to operate off of talking points, at least have the courtesy of disguising the fact a little bit. Especially if you're going to do it in consecutive posts.


    Apparently so, since you repeat this on a daily basis. However, if something walks like a duck, quacks like a duck, smells like a duck, etc.

    Yes, and their credibility is crumbling rapidly in the eyes of people paying attention.

    That's because they rather consistently leave glaring errors big enough to drive a truck through.
     

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