Invest in ME Conference 12: First Class in Every Way
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Further Reflection and Critique of the 4 Contamination Papers

Discussion in 'XMRV Research and Replication Studies' started by Megan, Dec 29, 2010.

  1. Megan

    Megan Senior Member

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    It has taken me a while to read those papers that came out last week. I am guessing there might be a few other in the same boat so I thought maybe there's room for another thread for people to critique the papers once they have read them.

    Having trouble getting my head around the Hue paper, but to get the ball rolling I have made a couple of observations about the Oakes and Robinson papers below.
     
  2. Megan

    Megan Senior Member

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    Does the Oakes paper results support the WPI Findings?

    Oakes et al tested 148 patients and found 21 were positive for XMRV/MLV by PCR (mostly controls).

    What is interesting is that, in addition to the much discussed IAP test, 19/21 contaminated samples were also identified as contaminated using the Switzer mtDNA test (table 1). Dr Mikovits has stated publicly many times that she tested all the WPI samples with this test and all came up negative. If this test can pick up 90% of contaminated PCR positives in the Oakes example then it seems inconceivable to me that if the WPI samples were contaminated that the WPI could have got a completely zero result on this test. On my reading the same PCR and mtDNA tests were used in both cases.

    Overall the mtDNA test still identified 30 of all the samples as contaminated in the Oakes paper as compared to 83 for the IAP test. The paper states that both the mtDNA test and the IAP are more sensitive than the PCR test.

    Obviously there will now be pressure on the WPI and Lo/Alter to run the IAP test on their samples. But in the absence of an IAP test, the above figures on the mtDNA test surely support the notion that the WPI or Lo/Alter studies were NOT contaminated?
     
  3. Megan

    Megan Senior Member

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    What are the correct mtDNA results for the Robinson Paper?

    After reading the Oakes paper, I went to look for the equivalent mtDNA figures in the Robinson paper and to my surprise found this paper seems to directly contradict itself on this point.

    They found 21 out of 437 prostate cancer cases to be XMRV positive by PCR (4.8%).

    In the results section of the abstract it states in relation to these 21 cases, “…many, but not all were positive for mtDNA.” Yet ‘Table 1’ in the back says 13/21 XMRV+ cases were not tested for mtDNA, one was positive and five were negative. The statement in the text directly contradicts what is in the table. There is no explanation given in the footnote of the table as to why the 13 samples were not tested (seems bizarre that they wouldn’t have been tested)

    Additionally the table still does not add up properly to 21 for the mtDNA test even if you allow for the missing 13. The ‘missing 13’ is really the ‘missing 15’. Given the contradictory statement in the text, this missing information seems suspect. If this paper didn’t test 13 of the samples, then why not?

    In any case, as with the Oakes paper, if they did find “many” XMRV+ samples positive using the mtDNA test then the same logic would have to apply, in that you would expect at least some “contaminated” samples should have shown up when Mikovits and Lo/Alter ran the mtDNA tests on their own samples.
     
  4. eric_s

    eric_s Senior Member

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    Good analysis, Megan, thanks.
     
  5. alex3619

    alex3619 Senior Member

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    Numbers Game

    Hi everyone, I want to run a quick numbers game regarding contamination. In this game I am going to presume that ALL of the positive controls are contamination: lets call them 5% of the controls (for simplicity) - and this could mean that every sample in Lo/Alter study is contamination too (not very likely, but give me a minute).

    If we presume that 5% of the original cohort by Lombardi were contamination, we get only 62% association. If we go with the revised 98% figure that is sometimes mentioned, we get 93%. Still very strong association, and now without healthy positives all positive people are sick. Hmmmmm ...

    Even if contamination is an issue, the whole argument still rests on patient samples being handled dramatically differently to controls.

    Bye
    Alex
     
  6. Mark

    Mark Former CEO

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    No time now, I will read this later...but taking the opportunity to raise a point I haven't seen mentioned...

    One of the papers mentions that the blood samples they used were left over from a 2005 study looking for correlation between CFS and (if memory serves) "a deltaretrovirus". Which raises some questions...

    What was that study? Was it published? Did we even know it happened? Why has that study and that retrovirus not popped up in discussions here before now? Did it happen behind closed doors? Was it a UK study? Was it bio ME research that we didn't know about? If so, what else has gone on behind the scenes? Why is it behind the scenes? And last but not least: if they were looking for a retrovirus association 5 years ago, why did they jump straight on the WPI and make arguments that a retrovirus doesn't fit the facts of CFS?

    I'm running ahead a bit I guess, since maybe that study was well-known and not what I'm thinking; perhaps it was an EBV thing or something. But I still have this feeling there might be something that was let slip there that could be worth exploring...
     
  7. August59

    August59 Daughters High School Graduation

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    Very good Mark! I have never heard of a study looking for a "deltaretrovirus". I sure would like to see the results of this study, but I can imagine that if it was done in the UK it was either found to be contamination or if results was positive it has been "sealed" til 2070(?). Partial sarcasm here!

    If those samples were left over from an earlier study could possibly mean they were contaminated during the first study. Are there other viruses that are real "deltaretroviruses"? Thanks!
     
  8. August59

    August59 Daughters High School Graduation

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    I second this! Thank you very much Megan!
     
  9. Sam Carter

    Sam Carter Guest

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    """"""""""""""""""""""""""""""""""""""
    In 2005, we initiated a study to examine the expression level of an endogenous human betaretrovirus, HERV-K18, in chronically ill CFS patients versus healthy controls.
    """"""""""""""""""""""""""""""""""""""
    (http://www.retrovirology.com/content/pdf/1742-4690-7-109.pdf)

    So make that a beta not a delta .... :D

    Prof Huber discussed the study -- which is ongoing, I believe -- at this year's Invest in ME conference.
     
  10. Sam Carter

    Sam Carter Guest

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    I can't tell how sarcy you're being here ... but I'll bite.

    Bovine leukemia virus is a deltaretrovirus. HTLV is a deltaretrovirus.

    The retrovirus Elaine DeFreitas may have found was a deltaretrovirus. Now remind me which country put a stop to that research ..... ? ;)
     
  11. Ecoclimber

    Ecoclimber Senior Member

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    Commentary & analysis on the four retoviology articles

    Contamination of clinical specimens with MLV-encoding nucleic acids:
    implications for XMRV and other candidate human retroviruses by Dr. Howard Smith pathology.

    http://www.retrovirology.com/content/pdf/1742-4690-7-112.pdf

    Makes for interesting discussion
     
  12. oceanblue

    oceanblue Guest

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    Great point
     
  13. oceanblue

    oceanblue Guest

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    Of Mice or Men?

    I think the issue of contamination by mouse DNA might be a bit of a Red Herring (think that's it for animal references...).

    Most of the Retrovirology papers merely showed that contamination by mouse DNA could give postive XMRV results, leading Virusmeister Ian Lipkin to comment on the importance of 'molecular hygiene'.

    However, the evidence that contamintion HAD actually taken place in previous studies was the Hue paper on phylogenetic trees. This work suggested the two WPI XMRV sequences from the original Science paper were so closely related to the XMRV in the human cell line 22Rv1 that the source of the WPI sequence was actually contamination by the 22Rv1 cell line.

    In other words, the contamination was by a human cell line cultured in labs, not from mouse DNA. If that's the case, any number of tests - IAP or mouse mtDNA - showing no mouse DNA contamination would be irrelevant.

    What would be really helpful now would be for WPI to show that such contamination by human cell lines, rather than mouse DNA, had been ruled out by their own internal testing.
     
  14. Angela Kennedy

    Angela Kennedy

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    Ok- still on the mouses- 'mouse DNA', that's NOT the same thing as XMRV, is it? XMRV is a retrovirus that infects mice (and possibly other animals)? Mouse DNA is the DNA of mice? XRMV doesn't have mouse DNA does it?
     
  15. alex3619

    alex3619 Senior Member

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    Hi Angela, no, but mouse DNA has something so similar to XMRV that it can be hard to tell the two apart, and some mice might actually carry XMRV, which is why all the debate. Nobody can yet explain why so few of the controls are positive if this is all contamination. Until they can, and can prove it, the contamination issue is just an hypothesis, which remains to be proven for the actual studies.

    Bye
    Alex
     
  16. oceanblue

    oceanblue Guest

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    Yes, mouse DNA is not the same as XMRV. But... Many mice have XMRV integrated into their DNA; XMRV started off life as a mouse retrovirus that lost the ability to reinfect mice cells but is still carried in the DNA of many mice. So if you have mouse DNA there's a decent chance it contains XMRV. Put another way, finding contaminating mouse DNA means that any XMRV detected may have come from mouse contamination, not the human patient. Still, not all mice carry XMRV so mouse dna contamination could in theory occur but not be responsible for postive XMRV findings.

    Correct, XMRV itself does not carry mouse DNA (well, it might just pick up tiny bits of it, but not enough to be detected by any of the mouse DNA tests discussed in the Retrovirology papers).

    However, my point was that the Hue paper suggested the WPI XMRV findings were contaminants from human cell lines, NOT mice.
     
  17. Megan

    Megan Senior Member

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    I take your point but those tests are not irrelevant if they are positive. There is one glaring omission in the Hue paper and that is that the Hue paper, and the others published in Retrovirology, emphasise the need to properly control for contamination. But where are the negative controls in the Hue paper? How do we know their reagents weren't contaminated? This would apply to bothe the testing they did on the 411 different cell lines as well as the analysis of the 22Rv1.

    Page 11 under the methods section says they were using:
    The Sato paper found an Invitrogen PCR product contaminated and therefore implicates the Lo/Alter sudy as contaminated because it used a different Invitrogen PCR product. Why not the Hue paper on the same basis?

    Also Hue et al did not run mtDNA tests or IAP tests on their samples, so how do they know they didn't get their own samples contaminated? I know they said a negative result on these tests would mean nothing, but surely a positive one might have meant the contamination might have come from somewhere other than the cell line itself?

    In short the other three papers published with the Hue paper appear to me to undermine the methodology of the Hue paper itself. Though, on reflection, it still might not undermine their genetic argument if the same genetic logic would apply to contamination from elsewhere?
     
  18. Megan

    Megan Senior Member

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    I am unable to follow the statistical analysis that the Hue paper has done but I think we need to be careful not to take this as gospel at this stage. People that understand this better than me on the other forum are saying that Baysean analysis is very subjective depending on what modelling assumptions are made. If anyone here has a better handle on it they might like to comment.

    I would note though that the Hue analysis doesn't touch the Lo/Alter finding. If they had included it, it would have wrecked their whole argument, so perhaps convenient for them that they left it out.

    It seems to me that the Hue andOakes paper did a good job on showing that the XMRV PCR test is not specific and can pick up other MLV's. But this is not exactly new news. It has been very clear since the Alter/Lo paper that the test can pick up more than just XMRV. As far as I understand it they used the Urisman/WPI tests as well, got a positive result and sequenced pMLVs from it. The WPI have made no secret of the fact that they have also found such variations in their sample since the publication of the Science paper.
     
  19. oceanblue

    oceanblue Guest

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    I haven't read the Hue paper for a while I don't have the energy now but what you're saying sounds like a fundamental critique of Hue's work.

    I did just find this under Methods
    but nothing at all on testing for mouse DNA contamination of their own samples.

    I think you're onto something very important here.
     
  20. Megan

    Megan Senior Member

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    Though I can't follow the statistics it seems important to me that the specificity of the testing for the 22Rv1 cell line would need to be the same as for the testing used in the studies, otherwise a narrower genetic range in the study examples could simply be a result of narrower range of specificity of the tests in those studies. I dont really understand what they did in the Hue paper to PCR test 22Rv1 cell line, but it seems important that this is looked at.

    It is noticable that the only published prostate cancer and CFS sequences of XMRV used in the Hue paper were almost all from the original Urisman 2006 paper and two from the WPI paper, and there are very few of them. Where did these come from? What type of testing picked these examples up? How specific were they?

    • The two WPI sequences were taken from a small subset (7/100) of the original Science cohort that tested positive for GAG and ENV on single round PCR. They therefore may not be representative of what was causing the nested GAG PCR to go positive for the rest of the 67%. The WPI have also openly said for some time that they have found other MLVs in their cohort since then.

    • The original Urisman paper is surprising also. Figure 1 and Table 1 indicates they tested 86 prostate cancer cases in total and found 9 positive using a combination of the virochip test and the nested gag PCR. 19/86 cases were tested using the virochip (Figure 1), leaving 67 remaing cases that were only tested by the nested GAG PCR. What is strange is that 8/9 positives were all within the subset of 19 that were tested on the virochip. Only one extra positive was picked up in the other 67 tested by PCR. Perhaps there is a reason for this, but it leaves the impression that they really needed the help of the virochip to identify the positives.

    In short, it seems to me that NONE of the XMRV prostate cancer or CFS sequences used in the Hue paper had been identified using the nested GAG PCR alone in either of the above studies. So the fact that the nested GAG PCR may not be specific to XMRV is irrelevant to the analysis.

    3) What is more obvious is that there have been many positive prostate and CFS papers reported since then, but with little more published in the way of sequences. This looks to me like most of the positive studies are yet to provide sequence data for their findings (including Dr Singh's prostate and CFS studies and those of Lo/Alter). Singh's public comment's about sequence diversity being the likly source of the testing differences and the differences found by Lo/Alter would indicate that there is a lot more to be revealed on this matter. It would therefore seem premature to be doing this type of genetic analysis before these investigations have been completed.
     

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