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New Paper - Gammaretroviruses - Maureen Hanson, David Bell

Discussion in 'XMRV Research and Replication Studies' started by VillageLife, May 21, 2012.

  1. VillageLife

    VillageLife Senior Member

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    Sensitivity of PCR Assays for Murine Gammaretroviruses and Mouse Contamination in Human Blood Samples


    http://www.plosone.org/article/info...seases (PLoS ONE Alerts: Infectious Diseases)

    Li Ling Lee1, Lin Lin1, David S. Bell2, Susan Levine3, Maureen R. Hanson1*

    Abstract
    Gammaretroviruses related to murine leukemia virus (MLV) have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs) or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.
     
  2. Bob

    Bob

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    Ah, the Maureen Hanson paper, at last!
    Thanks for posting it VillageLife.
    It's a nice long & complex paper for us all to read through!
    I'll try and look through it tomorrow.
    Will be interesting to find out exactly why she concluded that there wasn't any XMRV present in the blood.
    I'm also very interested in reading her conclusions re the antibodies.
     
  3. SOC

    SOC Senior Member

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    I haven't read the paper, yet, but I want to thank these researchers for making clear, accurate, scientific statements about their conclusions
    We've seen far too many conclusions lately that extrapolate far beyond the data -- the ones of the "We didn't find it, so it doesn't exist" ilk -- so it's refreshing to see sound research conclusions.
     
    currer and Bob like this.
  4. Mark

    Mark Acting CEO

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    Just read through this. It highlights what a complex and cutting-edge question this is; they used lots of different assays and did a whole lot of work, and they did initially find suggestive results with more positives in patients than controls, but then weren't able to reproduce those results using other assays, and in the end they weren't able to conclude they weren't contamination but also weren't able to identify the contamination, although they did find some evidence of some contamination.

    A couple of quotes...




    So they too found higher levels in patients than in controls. It looks like 50% of the 10 severely affected, 30% of 10 'recovered', and 15% of the 20 controls, with 1 severe and one recovered testing 'double positive'.

    But subsequent analysis generally failed to detect anything using many different methods, and they couldn't maintain this association they found initially.

    This is highly relevant to recent discussions:




    So they have a specific hypothesis here for systematic higher levels in patients vs controls, both in their own study and in Lombardi et al and Lo et al. But they weren't able to confirm their analysis in their own case.

    Interestingly, they propose a highly plausible theory for the different levels of detection in both Lombardi et al and Lo et al: in both, large batches of patient and control samples were assayed at different times. So different levels of reagent or environmental contamination at the time of the assays are a possible explanation.

    I think this is interesting because it is a different hypothesis to "patient samples were handled more frequently", which I never bought. This hypothesis is more subtle than that: it is the potential for different levels of environmental contamination at a stage in the process that nobody previously suspected might be subject to contamination. This is significant because it emphasises that whatever 'mistakes' may have been made, there were not obvious risks to anybody before these experiments - that removes the burden of blame placed on Dr Mikovits by some.

     
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  5. Mark

    Mark Acting CEO

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    This is important because it is only fair to emphasise again that nobody knew about these risks before Lombardi et al, in that the levels of contamination of lab products with XMRV and related MLVs are now extremely widespread. That's significant because again it shows that Dr Mikovits could not have been expected to expect this to be the case - it is a shock to everyone. It's an even more significant point, IMO, because the completely unexpected and unknown spread, which has been going on for 15 years or more, ofthis particular lab-created retrovirus that infects human cells, is a big surprise finding, and it is surely an important one - and should be bigger news. How much more of this has been going on? And has any such agent, ever in history, infected anything which has been distributed for human use (such as specific cell lines used to grow vaccines)? The scale of this problem and the length of time it has been occurring, by the law of averages suggests: Almost certainly, yes. No pathogenic example has yet been identified, but that is not to say that one does not exist. Sporadic cases of H1N1-induced novel forms of narcolepsy, anyone?




    Two points I've recently highlighted elsewhere on these forums are re-iterated again here.

    It is still unknown whether unkown retroviruses are inciting factors in ME/CFS. Obviously, but seems to need stating.

    Most of the studies of ME/CFS samples have not used primers that would detect all groups of MLVs. So MLVs, other than XMRV specifically, have not been much searched for.

    And as a final comment: after all this time, after all the analysis in this study, all the experiments performed, it still was not possible to nail the question of whether the sequences they sporadically detected really did come from the patients' blood, or not. That just illustrates how cutting-edge and difficult the science of this is, and shows that there is still a great deal of work to be done before all the mysteries around these viruses will be fully explained.
     
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  6. Mark

    Mark Acting CEO

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    Absolutely - I couldn't agree more: it is just so refreshing to see clear, accurate, scientific statements that don't overreach.

    When that is not the case, it is clearly visible to even non-experts such as ourselves, and this engendered an enormous amount of quite legitimate distrust of many researchers in the early days. When you can clearly see a scientist's bias on display, from the illogicality of their conclusions which are not just not remotely justified by their own findings, on points of simple logic alone, that is a very frightening thing to see.

    Reading a paper like this one, where the language is sober, logical, reasonable, and fair, and the conclusions are justified, is such a massive breath of fresh air that it really takes my breath away. I do hope that this sort of thing is the norm in science, and that what we see regularly from the likes of White and Wessely et al, and what we saw from McClure and many other early researchers and commentators on XMRV, is very much the exception.
     
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  7. Mark

    Mark Acting CEO

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    One other point I meant to add, which seems to me quite crucial.

    Although there have now been many published studies (I can count at least 7, I think there are more), in both ME/CFS and prostate cancer, which have reported statistically significant higher levels of XMRV/MLV detection in patients vs controls, I have yet to hear of a single example of a study or even part of a study which found more XMRV/MLVs in controls rather than patients.

    Now: while the explanations of differential handling of samples do make sense, there are now quite enough results to make it seem most peculiar that this is a one-way street.

    This could potentially be explained by many studies having been done which did find much more XMRV or MLVs in controls than in patients, but which were not published due to embarrassment. But it seems most odd that anyone should not publish in this circumstance, because such a study would massively affect the debate and would be very significant. I doubt this is the case, but if anyone has evidence of a counter-example, let them come forward...

    If we approach this question on the balance of probabilities: The odds of flipping a coin and getting heads 8 times in a row are one in 128.

    So we might reasonably conclude that it is more than 99% probable that the unexplained discrepancies are something specifically to do with the patient samples themselves, and not due to random contamination of batches at the time when the assay was performed.
     
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  8. alex3619

    alex3619 Senior Member

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    Hi Mark, this is the crux of why I do not buy the counter-claims as proved. They might be right, but until questions like this are answered they are not "proved" right. What has happened though is the standard of evidence to justify the XMRV hypothesis has been raised substantially, so high perhaps that it will be hard to justify the hypothesis even if it is correct.

    Pop quiz: how long did it take the hypothesis that H. pylori caused peptic ulcers to be accepted? Read the answer in my new blog due out soon - others have said it before, but not many seem aware of the actual facts. In case anyone is looking at the Wikipedia, its very very wrong.

    Bye, Alex
     
  9. currer

    currer Senior Member

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    Tissue sampling anybody?
    Time for a different approach. The problems with blood samples seem insurmountable

    On another note, it is scarey how all the laboratory materials are contaminated with mouse DNA.
    If this contamination is impossible to eradicate, the human population has certainly been exposed to it." Sterile" takes on a new meaning.
    .
     
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  10. heapsreal

    heapsreal iherb 10% discount code OPA989,

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    bond uni i have heard are applying for permission to do spinal tap and testing spinal fluid in a possible future cfs study
     
    Bob likes this.
  11. alex3619

    alex3619 Senior Member

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    Ouch heaps, should I remove my name from their list? :eek:
     
  12. currer

    currer Senior Member

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    I've just thought - have Hanson and Bell taken on board the recent discovery by Maureen O'Keefe that commonly used PCR reagents inhibit the reproduction of MRV's? Remember that one?
    Could this explain the diminishing positives with each round of testing?
    http://okeefe-lab.blogspot.co.uk/p/current-lab-projects.html
    Can anyone recover the link to that report on her blog? I'm too busy to look atm.
     
    Bob likes this.
  13. Mark

    Mark Acting CEO

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    Wow, I like that currer, I missed that one. It also seemed odd to me that out of all the different tests they ran, the only one that found results was the very first one. Bit of an odd coincidence.

    Now wouldn't that be a thing: if reagents you were using progressively suppressed replication, the more pre-test calibration you did before running the tests proper, the less chance you would find anything - a potential explanation for negative results, and it'd almost be like the more carefully and rigorously you prepare and the harder you try, the less likely you are to find anything at all. That appeals to my sense of irony...:)
     
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  14. natasa778

    natasa778 Senior Member

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    currer likes this.
  15. Sam Carter

    Sam Carter Guest

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    I've just skim read the paper so I may have misunderstood things, but they do say:

    "Nevertheless, we are confident that negative results are not due to PCR inhibition because we regularly observed non-specific amplified fragments of variable sizes."

    ETA: They also write: "While one group used the dUTP-UDG method to prevent carryover of PCR products [20], our preliminary experiments suggested that this technique reduces the sensitivity of our PCR assays, which is consistent with a subsequent report [21]. We chose instead to reduce the possible environmental contamination of PCR assays by developing a single-round assay that can detect spiked plasmid DNA with the same sensitivity as the previously described gagO/gagI nested PCR."

    Reference 21 is Bacich DJ, Sobek KM, Cummings JL, Atwood AA, O'Keefe DS (2011) False negative results from using common PCR reagents. BMC Res Notes 4: 457.
     
  16. natasa778

    natasa778 Senior Member

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    This bit is also worth quoting. Great thinking currer!


     
  17. natasa778

    natasa778 Senior Member

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    This is what O'Keefe paper is suggesting, not sure if Hanson study took these steps

    Also we are not talking about total PCR inhibition here, but rather as Mark put it "the harder you try, the less likely you are to find anything at all". At what stage/s were those fragments amplified, and does the 'strength' of PCR (the degree of inhibition) have effect on WHAT is amplified?
     
  18. natasa778

    natasa778 Senior Member

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    ok Hanson paper address - at least seems to - the PCR inhibition problem identified by O'Keefe:


    Anyone?

    Does this come anywhere close to possibly explaning the difference in rates of detection btw patients and controls?
     
  19. Sam Carter

    Sam Carter Guest

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    Perhaps you could write this up as a comment / question, Nastasa, and get a definitive answer from the authors? I think it would be useful to hear in more detail what steps they took to avoid the problems highlighted by Dr. O'Keefe.
     
  20. Bob

    Bob

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    So that does directly address the O'Keefe paper doesn't it.

    From my memory (which isn't reliable), of reading the O'Keefe paper, single round PCR can be affected if the PCR slide has been used before, either in the manufacturers' or the researchers' calibration, or if a negative slide is reused. If there is even a tiny amount of contamination, then the results can be false-negative. (I might have some of this wrong though - my memory is notoriously useless!)

    Talking about XMRV research in the 'discussion and conclusions', the O'Keefe paper states:
    "Therefore, amplification of the high copy number positive control could occur while samples positive for a low copy number virus could be inhibited by carry-over PCR contamination."
     

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