DNA Extraction Columns Contaminated with Murine Sequences

This paper makes me very thankful for two things:

1– Bossman is extremely neurotic about controls (this apparently, is inheritable, because I am too).
2– I dont work on a mouse virus.

If the list of potential sources of contamination in the XMRV–>prostate cancer, XMRV–>CFS stories werent long enough, here is yet another source of contamination to add to the list:

In assessing the prevalence of XMRV in prostate cancer tissue samples we discovered that eluates from naïve DNA purification columns, when subjected to PCR with primers designed to detect genomic mouse DNA contamination, occasionally gave rise to amplification products. Further PCR analysis, using primers to detect XMRV, revealed sequences derived from XMRV and pMLVs from mouse and human DNA and DNA of unspecified origin. Thus, DNA purification columns can present problems when used to detect minute amounts of DNA targets by highly sensitive amplification techniques.

DNA purification columns are ubiquitous. They are little columns with a membrane in them that DNA ‘sticks’ to, so you can wash all the garbage (proteins, extra free nucleotides, enzymes, etc) off, and then when youre done washing the DNA, you add water and the column so the DNA doesnt stick to the membrane anymore, and youve got pure DNA in water. Hurray! Purifying DNA with a kit with these kinds of columns is much faster than the old-school protocols (though we often use the old protocols because they are cheaper and you can get a higher yield, but I digress). So people use these kinds of kits to purify DNA from bacterial preps, to purify DNA from agarose or acrylamide gels, to purify DNA from PCR reactions, to purify DNA from mammalian cells, to purify genomic DNA from viruses– everyone uses these kits. Particularly, everyone uses Qiagen brand kits (we often use off-brand, again because they are cheaper, but if you want to be 100% sure of a finding, you use the Qiagen).

But apparently, because moosey mice are everywhere they are contaminating everything, including the ‘DNA purification columns’ in Qiagen kits. So if you are looking for ‘mouse DNA’, or in this case, ‘the genome of a mouse retrovirus that is present in mouse DNA but youre trying to say its in human samples’, you are screwed 11 times for every 68 columns. The contaminant? Same sequence as VP62.

There is one way around this contamination– Qiagens SUPER SPECIAL columns:

It is worth noting that QIAamp Ultraclean Production (UCP) Pathogen columns which are certified to be free of contaminating microbial DNA yielded no amplification product for IAP or MLV-related sequences in 50 “mock-extracted” columns.

Those kinds of columns are usually reserved for very specific protocols (they are ~twice the price of the regular columns, which ~twice the price of off-brand), but if you do mouse research, you might just have to use these super special columns all the time.

Or, you can go back and use old-school protocols and make all your solutions in-house.

But they might be contaminated too.

:-/

Very glad I dont do mouse research.

And just to reiterate, until the dozens and dozens and dozens of contamination issues are settled with XMRV (ignoring all of the cell bio, which implies XMRV cannot productively infect human PBMC. ignoring the animal model with implies XMRV doesnt do anything. ignoring the epidemiology, or rather, the lack of epidemiology regarding XMRV ‘infections’), the contamination issue is basic, basic science that needs to be dealt with before anyone can speak a word in support ‘XMRV IZ INFECTIN DEH PEOPLES OMFG!!!!’

Again, XMRV is not infectin deh peoples.

Also, poor Carl Zimmer tried to write about the threats researchers (and journalists, and bloggers) have gotten from the nutty XMRV–>CFS proponents. Aww :(

Comments

  1. #1 JohnV
    August 22, 2011

    haha @ the comments on Carl’s blog.

    “34. Zac Says:
    August 22nd, 2011 at 6:53 am

    I don’t blame anyone for fighting for their freedom.

    Look at Libya and Egypt. The ME/CFSers have been suppressed for decades. It’s time to push for solutions.”

  2. #2 JohnV
    August 22, 2011

    That’s particularly amusing considering how many comments before it were like “omg no one with CFS could ever be a threat, we’re all sickly and bed ridden” and then that guy is like “omg time for armed revolt”.

  3. #3 David
    August 22, 2011

    Thank you for your continued enlightenment regarding the XMRV research.
    I do wish you would see the ‘nutty XMRV –>CFS’ issue as perpendicular to this though. I remember a lovely sunny day reading the first headlines on this issue. Momentarily years of anxiety and fear lifted as I thought the unthinkable – my trauma as a carer might be over.

    Until you suffer from a something so catastophic that it ruins your life, puts an end to your PhD and dreams of success maybe – it is impossible to empathise with the way people hang on to science news with baited breath.

    People should back off maybe from thier ‘Lorenzo’s Oil’ attempts to find out what is going on, and nasty emails are a no no of course – but do not get dragged in with siding with a group of people who want to dismiss an entire patient group as nutters.

    Some research on the history of AIDS would serve you well – sorry, that was cheeky, of course you know this history, and you imagine you would not have seen the antics of a group of gay rights extremists as having any bearing on scientific reality.

    It is actually very sad that this has panned out this way, and CFS patients are having to cope with the double misery of scientists messing with thier heads, as well as uninformed journalists that are painting them to be all crazy ill deserving people anyhow.

    I know you are not a doctor, but still the basic oath ‘do no harm’ is a good jedi scientist code to live by.

  4. #4 Johan
    August 22, 2011

    @David
    When I look at the XMRV-hype I think most of the messing with the heads is done by patients themselves.

    If I try to visualize what needs to be done to raise awareness for ME/CFS in my own country, one of the things is to start apologizing.
    Apologize to the publisher of 2 of the main newspapers who nearly got sued for slander thanks to a self-professed CFS advocate and who were at the receiving end of an email campaign. Apologize to the Minister of Health who got declared persona non grata by the most active patient organization. It beats me what a patient organization hopes to achieve after that. Apologize to …
    But first and foremost find a way to prevent shooting ourselves in the foot over and over again. And I have no idea where to begin.

    PS: Yes, the articles about the threats are an example of sloppy reporting. Did the journals check the facts? Did they ask patients or patient organizations what they thought of the actions of those individuals? If you look at the comments on Carl Zimmer’s blog and on the newspaper articles you will see that there are plenty of patients who condemn those threats.

  5. #5 DrDuke
    August 22, 2011

    The sequences from the column contamination were not identical to the VP62 clone of XMRV, they were just somewhat similar. They more likely come from mouse DNA, the pre-XMRV1, preXMRV2 and other endogenous MuLVs, than from XMRV.

  6. #6 HFM
    August 22, 2011

    Did they check for human sequences too? I’d bet that there’s more stray bits of human than stray bits of mouse in the average Qiagen packing facility…though I realize the XMRV wouldn’t have come from the people.

    Makes me even more glad I don’t work with Sooper Sekrit, no really it’s in there, cryptic sequences / figments of lab imagination.

  7. #7 Smynn
    August 23, 2011

    If you really believed that contamination is everywhere and in everything you would,

    A. Not use PCR anymore
    B. Dismiss HTLV and HIV as contamination
    C. Be adamant it would have been passes into human.

    As you can see they are contradictory beliefs. Perhaps if you realised there is no scientific evidence of contamination you would understand how illogical your feelings are. Come back when you have at least 10 years under your belt.

  8. #8 Smynn
    August 23, 2011

    If you really believed that contamination is everywhere and in everything you would,

    A. Not use PCR anymore
    B. Dismiss HTLV and HIV as contamination
    C. Be adamant it would have been passed into humans.

    As you can see they are contradictory beliefs. Perhaps if you realised there is no scientific evidence of contamination you would understand how illogical your feelings are. Come back when you have at least 10 years under your belt.

  9. #9 Jonnfg
    August 23, 2011

    No mouse contamination, in any if these papers looking to find mouse contamination, has been shown to be any of the strains or variants found in people. Isn’t that fascinating! And 22Rv1 appears to have been contaminated by VP62. Again, isn’t that fascinating!

  10. #10 Mary
    August 23, 2011

    @7-8 Are you embarrassed? No? If I have read your post correctly then I am embarrassed for you..if I’ve read it wrong..I’ll apologize..but I believe there is widespread contamination in labs, therefore, according to you, I need to grow up and I am illogical?

    @Johan..I’ve followed your personal story in the XMRV debate..Do you get hate mail?

    Most of the comments at carl’s = facepalm..somehow, these threats are OK cuz they are ill, frustrated, and misunderstood?

  11. #11 Johan
    August 23, 2011

    @Mary Occasionally, just hate, no threats.

  12. #12 RRM
    August 23, 2011

    New negative paper:

    http://www.hindawi.com/journals/av/aip/268214/

    This time I’ll have to agree with the forums if they call this paper a dsigrace though. How can John Coffin be involved with such tripe (even though it’s provisional):

    “The Multicenter AIDS Cohort Study (MACS) provides an opportunity to assess the prevalence of XMRV infection and it’s association with HIV-1 infection among men who have sex with men.”

    Perhaps they should join Abbie and do away with apostrophes altogether….

  13. #13 ErkLR
    August 23, 2011

    Looking at my Qiagen RNA columns, I see they are not gamma-irradiated. Maybe they should add that to their production process for all their columns. But I suppose considering it’s all automated, it’s probably a huge expense to add that at the end.

  14. #14 Dr. Lavarian
    August 23, 2011

    Seems bizarre that Qiagen brand kits more scientifically acceptable than off-label DNA Kits and that they would be the only providers of microbial-free columns.

  15. #15 peeps
    August 24, 2011

    I agree with Smynn. How, if contamination is widespread and has not been known about, although I find that hard to believe, can the virus and other viruses not have found their way into humans?

  16. #16 Charl
    August 24, 2011

    Peeps – firstly, it’s only been in the last few years that viral arrays (which were used to discover XMRV) and high-throughput sequencing (to detect trace amounts of DNA that Sanger sequencing might otherwise miss) have been available and affordable enough for this kind of investigation to go ahead. Secondly – scraps of DNA =/= productive, replication competent virus or viral infection. There are probably loads more viruses like XMRV out there (not causing disease, not being noticed) that we will detect more frequently in the next few years (eg mouse mammary tumour virus, TT virus).

    You come into contact with lots of viruses, all the time, from lots of different sources. Only a small proportion will successfully infect you, and a smaller proportion still will manage to achieve long-term infection. And a smaller proportion still than that will cause disease in some people (eg HPV, EBV, herpes simplex, HIV, HTLV).

  17. #17 Jack
    August 27, 2011

    Morning chaps,

    Would anyone care to comment/explain the methodology and results from this study by Katzourakis earlier this month?

    http://jvi.asm.org/cgi/content/abstract/JVI.00827-11v1

    Can such a study be only deemed ‘hypothetical’ for instance?

    Really struggling to interpret it and from what I have tried to read elsewhere – others (non-professionals) are to.

  18. #18 Mar
    August 27, 2011

    @10 Mary? but replies as if they were ERV

    Either the argument is that human gammas are everywhere and will jump to anything in a lab at any time, so cannot have been prevented from getting into human biologicals, or they are not, which is the case. And now you have to provide scientific evidence for contamination.

    As Lo er al. according to Towers found xenotropic sequences as well as polytropic and modified polytropic and Lombardi et al. have xenotropic polytropic. They have confirmed the finding of each other.

    “CFS type 1 infected patients are derived from three strongly supported and distinct regions of the tree, namely, the polytropic, modified polytropic and xenotropic clades.”

    “Another 2010 (MLV_001_2010) sequence is robustly placed within the xenotropic clade, at the base of the XMRV cluster.”

    http://jvi.asm.org/cgi/content/abstract/JVI.00827-11v1

    @Jack
    Read this explanation, it is informed and qualified.
    http://www.imeassoc.com/Katzourakis_et_al.html

  19. #19 RRM
    August 27, 2011

    That explanation is neither informed nor qualified. It’s still a good example of motivated reasoning though, so I am glad you have posted it. A good rule of thumb is that you should not trust any “research group” that blows not one but two chances of saying “Towers’ paper” correctly.

    Jack, phylogenetic analysis is considered to be very robust. It can be easily experimentally tested and this has of course been done numerous times in the past. That is exactly why it is considered to be robust instead of some untested little hypothetical theory. More importantly, in the crazy event that everything that scientists think to know about phylogenetics would magically not apply to XMRV/MLV-like sequences, this could be very easily be proven by the proponents of this hypothesis. Simply grow some XMRV/MLV-like sequences in the lab, take some samples now and then and show that phylogentical analysis does not explain the reality of your longitudinal sequences.

    They can’t do this, while there are grand prizes and large sums of money to be won.

    Now, I readily admit that (like the people who made up that stuff in the link above) I know little of phylogenetics. However, because Abbie isn’t responding, I can try to give you some basic idea what this analysis is about.

    Suppose I inject you with a “virus” that consists of the following five nucleotide sequence:

    aaaaa (virus 1)

    Now, some time later, someone gives me two “Jack” samples that were taken at different times after that. They are:

    caaag (virus 2)
    aaaag (3)

    Which of these two sequences would most likely be ancestral to the other one, given the fact that we know virus 1 is ancestral to these two? (I’ll disregard the negligible chance that these sequences reflect independent infections BTW.)

    First it’s important to note that there are really three possibilites: virus 2 could be ancestral to (3), or (3) to (2), but it is also possible that both sequences would have formed directly and independently from (1).

    [Preview was messed so posting in two parts. See below]

  20. #20 RRM
    August 27, 2011

    [continued]

    In this case I think it’s pretty easy to see that the most likely explanation is (1)-(3)-(2). Why? Consider the a at nucleotide position 1 of virus 1 (and virus 3). If (2) would be ancestral to (3), that nucleotide would have to have changed from a to c and then back to a again. If all of the nucleotides had the same chance of changing, there would be a <5% chance of this happing. Likewise, if (2) and (3) had independently derived from (1), they would both have accidentally changed the a at nucleotide position 5 to g. The chance of that happeing would be the same.

    The changes in the most likely tree, (1)-(3)-(2) wouldn’t have to rely on such coincidences. Of course, with such a small sequence and so little sequences, the conclusions above are definitely not robust. It would be unlikely, but these changes could still be reasonably explained away by chance. However, when you have 350 bit sequences and uncountable other sequences to compare the with, and you have multiple nucleotide changes, the conclusion will get very convincing.

    In the case of the Lo sequences, these scientists showed that the sequences in the samples that were taken from the same patients, were highly unlikely to be ancestral to the sequences in the samples that were taken from these same patients several years later. They did this by checking the changes at different nucleotide positions and by comparing these with known mouse viruses.

    Though unrelated, I think this also shows how the forums are terribly wrong about phylogetic trees perhaps not applying to HTLV-1 like viruses or arguments like that. Phylogentic analysis is (to my knowledge) not dependent to the sort of virus you are investigating. The computer program will not “know” whether you have entered an HIV or an HTLV-1 sequence. It will just analyze the nucleotide differences that are within the sequences entered. Of course, when there is little variation it will be more difficult to draw conclusions, but the important thing is that the analysis takes that into account. If there is not enough diversity to create a tree, a different tree will be created every time, and if the same tree is drawn ~100% of 100,000 times, the diversity is apparently sufficient.

    Remember, if these scientists would be really tweaking the parameters of their computer programs to get the outcomes that they want, it would be VERY EASY to demonstrate that. Phylogenetic computer programs are freely availabe everywhere, and if the “authors” of that laughable piece of tripe would be actually “qualified”, they would not merely insinuate such things but DEMONSTRATE that one could reasonbly get to other conclusions using one of these many computer programs.

  21. #21 Jack
    August 27, 2011

    Thanks for that :)

  22. #22 Benoni
    August 27, 2011

    From the imeassoc link: “Using this test (Shimodaira Hasegawa) when one of two typologies to be compared is one of maximum likelihood and the typologies have not been determined a prior completely invalidates the mathematical assumptions that underpin this test.”

    What a lying shit. That statement applies to the Kishino-Hasegawa test. The Shimodaira-Hasegawa test was specifically designed to get around that problem. Makes. me. so. angry.

  23. #23 Benoni
    August 27, 2011

    Also, LOL@”typologies”. Twice! What a total fucking retard.

  24. #24 ERV
    August 27, 2011

    Either the argument is that human gammas are everywhere and will jump to anything in a lab at any time…

    ‘Human gammas’ (lol, wut?) are not everywhere.

    Mice are everywhere (along with their associated hair/dander/poop–>DNA–>ERVs that could appear to be exogenous ‘retroviruses’ to someone not looking for contamination), and mouse retroviruses are contaminating all our cell lines, and mouse products are used for the generation of standard laboratory reagents (again, providing mouse DNA–>ERVs that could appear to be exogenous ‘retroviruses’ to someone not looking for contamination).

    You are on ScinceBlogs. If you dont understand something, ask. We love questions. However, when you aggressively make incorrect assertions, you just end up looking like an asshole.

  25. #25 David
    August 31, 2011

    Is this

    http://projectreporter.nih.gov/project_info_description.cfm?aid=8178650&icde=0

    a waste of a quater of a million dollars? Genuinely wondering why after the Science retraction debacle, plenty papers going ahead. Inertia?

  26. #26 Mar
    September 1, 2011

    @ERV

    Then you now admit that whether mouse contamination is everywhere is nothing to do with human gammas and that there has been more than ample opportunity for further human gammas to have been created due to the high levels of mouse contamination that some claim they knew nothing about.

    Coffin may wish to claim a virus was created in a cell line without providing the evidence to back up that claim, but he is now saying there is also another virus in a separate cell line, which turns his hypothetical rare event into something much more expected. None of which stop those viruses being present in humans.

    As you will know, all positive xenotropic polytropic and modified polytropic studies have looked for mouse contamination and found non. This has also been supported with further experiments.

  27. #27 Mar
    September 1, 2011

    Are you aware that John Coffin has detected XMRV in HIV patients and in prostate cancer patients? HIV does not appear to be associated with low levels of detection, around 3%. However, Prostate cancer is again indicating an association, around 12% this time.

  28. #28 RRM
    September 1, 2011

    “None of which stop those viruses being present in humans”

    I take it you do believe in time travel?

    “all positive xenotropic polytropic and modified polytropic studies have looked for mouse contamination and found non[e].”

    First, you would not expect mouse contamination with what Lombardi et al. found (XMRV). Remember, the evidence indicates that XMRV is a human cell line contaminant.* Specifically, all bona fide XMRV sequences from published studies are thought to be direct or indirect contamination from 22Rv1. Not finding mouse DNA is ENTIRELY CONSISTENT with that hypothesis, as 22Rv1 (or VP62 or any other indirect 22Rv1 derived XMRV) is NEGATIVE for mouse DNA.

    Second. Lo et al. DID NOT test for mouse DNA using the IAP assay. Yes, Lo reported in December of 2010 that he did test using the IAP assay later on, but he also reported that the assay was actually LESS SENSITIVE than the test for mitochondrial DNA in his hands. This indicates that he did something wrong with the IAP assay, as all other reports have concluded that the IAP assay is way, way more sensitive. Hanson’s results have not been published (and I think it is getting more and more unlikely that they will) and until they are and she shows the sensitivity of her IAP test, it’s premature to conclude anyting about it.

    Finally, because we are dealing with trace amounts of contamination that sometimes require weeks of culturing to detect (Hanson also cultured her samples before doing PCR), it can (rather ironically) mean that this is why positive labs are not detecting contamination. Also, with regard to culturing of samples it should really be made perfectly clear when the contamination tests were being done: before, or after culturing of samples. Of course, the latter is the only way to go in this case.

    (*Even though you probably won’t agree, you should always be able to provide evidence AGAINST an hypothesis you wish to falsify)

  29. #29 Put
    September 2, 2011

    Yet you are missing a crucial piece of information called evidence. There is nothing that shows contamination has occurred. More than likely 22Rv1 is infected with VP62. That is what we are really seeing. It probably happened when Miller looked at the cells in 2009.

    There is no reason to believe the IAP assay is more sensitive than the mtDNA assay. It it a matter of what you assess your assay in. Do you know the difference? I doubt it.

    Why are you mentioning Hanson?

    You will know that you cannot have an immune response to a contaminant. The Lombardi serology assay can only detect an MLV virus, not a engodgenous human or mouse MLV. You will also know that it is possible to do everything possible to control for contamination, but without evidence you cannot assume it has occurred. You would have to test that hypothesis, which has not happened.

    Anyway, Coffin is detecting XMRV using certain assays, not optimized for humans, thereby confirming the virus in humans and Towers has confirmed Lo et al found the same virus.

  30. #30 RRM
    September 2, 2011

    “There’s no evidence”? Please stop repeating the silly forums’ memes. There is plenty of evidence that indicates that Lombardi et al. and Lo et al. were contamination. In fact, the level of evidence presented against these findings would be unprecedented for a “true” finding. Requiring “evidence” does not mean that Coffin must acquire CCTV footage of mice crawling around Lo’s lab or something like that. Could you give me one single example of an independent experiment by an independent scientist that could produce (your definition of) “evidence” of contamination?

    There’s every reason to believe that the IAP assay is more sensitive: every published study that investigated the sensitivity of the IAP assay reported that is was way more sensitive.

    As for the immune response: no study, not one, ever in the history or the future, can be regarded as conclusive evidence in favor of any given hypothesis. Results should be reliably reproducible. We will see if the serology results hold up shortly in the patients that Ruscetti/Mikovits/Lo have chosen themselves using assays that they have chosen themselves. I cannot wait for the next “explanations” if their initial results again fail to hold up.

    Coffin is not “detecting” XMRV in human samples. I know you don’t want to believe this and think that positives are always true positives but negatives are not always truly negative. However, as Abbie herself has pointed out several times on this blog, there is always the possibility of reporting false positive. If this is hard for you to believe, just look at the (“public”) experiments Mikovits has performed after the Science study. Both times (Phase i and IIB of the BWG) she reported a false positive on a negative she herself (and Ruscetti) had pedigreed as being negative by multiple, independent tests. On another occasion, she detected XMRV in 1 out of 3 healthy controls, which would require quite a bit of “bad luck” (the odds of this happening when you yourself are only able to detect XMRV in ~4% of controls is about 11%).

    Ruscetti also reported a false positive on the well pedigreed negative sample BTW in Phase IIb. To get back to the serology argument: how can a sample that is well pedigreed for not containing any XMRV/MLV-like viruses nor any sort of immune response to XMRV/MLV-like viruses, still have an immune response to that non-existing virus in blinded testing? I can tell you that by far the most likely explanation is that this result was not truly an immune response to any MLV related virus but just an artifact of some kind. I am truly sorry though that without a video camera and access to Ruscetti’s lab, I don’t have any evidence of this by your definition of “evidence”.

    The BWG results are due for release shortly. Let’s see if Mikovits/Ruscetti/Lo can at least reliably differentiate between supposed positives and supposed negatives. That really seems to be the only possibility that can turn this thing around at this moment.

  31. #31 David
    September 2, 2011

    Yeah, this and plenty more papers are on the way.
    Science may have suggested a retraction, but as yet nobody has said “OK – stop people – this is a contaminant – put your dishes down”, so there are plenty of projects proceeding on wrong / right assumptions as to what this thing is.

    So better just to wait and see. The truth will out – but not on endless nanoscopic forum discussions on methodolgy. It won’t be outed any quicker either by sensationalist blog headlines – “arrghh it’s everywhere – get it off me….”

  32. #32 Jack
    September 3, 2011

    Following on from Carl Zimmer and the Guardian newspaper piece ‘pinch of salt’ though it might have been…

    I see that Steven Novella has written a very interesting piece about the trend (perhaps) being evidenced by those who seem bent on following a singular course of action and not accepting of evidence or bending their fervency to it:

    http://theness.com/neurologicablog/index.php/science-by-intimidation/

  33. #33 Shotman
    September 3, 2011

    I agree with Dave, but the devil is in the details and that is where you find assays do and don’t work.

    RRM, have a go at reading the papers.

    Both papers admit to setting a new standard for what is counted as a positive. Coffin has shown the virus is in people. So any 00 study is suspect for not detecting a background level.

    “if we had based XMRV infection on a single diagnostic method (either PCR or serology), the apparent XMRV prevalence would be 1.2%; 0.3% (1/332) by PCR and 0.9% (3/332) by serology. ”
    http://www.hindawi.com/journals/av/aip/268214/

  34. #34 David
    September 3, 2011

    This…

    ‘specimens….were obtained from the Whittermore-Peterson Institutes’ sample repository’

    from this…

    http://merutt.files.wordpress.com/2011/05/mikovits_lombardi-publikasjon-mai2001.pdf

    Do you think they should clear out the fridge then?

  35. #35 RRM
    September 4, 2011

    Good read, Jack, thanks for that.

    @David.

    That really does suggest that WPI should clear the fridge. It’s not that 118 “XMRV positive” patients were selected for this study. No, they selected 118 CFS patients from the Lake Tahoe cohort for this study. Only after they found this distinct inflammatory signature during their fishing expedition, they chose to test these patients’ blood for XMRV, found all 118 of them positive and only then they reframed their findings.

    Besides this being methodologically stupid, it is also a very strange “finding” in the midst of this “oh, XMRV is so hard to find in blood” controversy. Remember, WPI were asked to provide their own XMRV positives for the Blood Working Group study. When you can readily detect XMRV in 118 out of 118 patients in your lab but later fail to discriminate between your self chosen positives and your self chosen negative(s) better than mere chance would predict, it is indeed time to clear the freakin’ fridge.

  36. #36 David
    September 4, 2011

    @RRM

    I’m confused by this paper. It reads to me like they just took phials already marked XMRV+ or whatever from the sample bank. No actual further XMRV testing was done.

    Using some form of cluster analysis was done – which as everyone knows given enough variables you can differentiate any group – that is err – already differentiated. Remember that XMRV or not these people were ill for some reason, some with other things , eg herpes, EBV, so it would be surprising if interlukin/cytokine regulation could not be used as variables in some cluster model.

    It is a nice idea, the work anyhow, similar multivariate marker research is a good idea, but NO mention of any of the negative XMRV work. Why would In Vivo not mention this while in review, it’s not a vanity journal?

    I’ll re-read it – again!

  37. #37 genie
    September 5, 2011

    @RRM

    Only a fraction of patients in Lombardi were from the outbreak. Read the response from Mikovits and Ruscetti. There were also 101 patients in the study not 118.

    XMRV is one variant of gammaretrovirus. Gammas along with other retroviruses like Delta’s are rarely found in the blood. Have you ever heard of the monkey studies where they clearly show the same pattern, where the virus leaves the blood and settles in the organs.

    Coffin is now detecting prostate cancer positives and HIV positives, or what is more of a general population level. This strongly indicates there should be no studies finding nothing.

    @DAvid

    You cannot have negative Canadian criteria ME patients when the virus is now found in 99/101 original patients. Either you have a cohort of ME patients who are infected with HGRVs or you don’t have ME patients.

  38. #38 ERV
    September 5, 2011

    Two years of this shit, and ‘CFS patients’ havent learned a goddamn thing.

    Two fucking years of saying the same thing over and over and over and over…

    Like fucking Creationists.

  39. #39 POL
    September 5, 2011

    ERV do you know anything yet about gammaretroviruses? They high recombination and low mutation rates. There preference for tissue and not blood? Why are you incapable of only seeing the world of retrovirology through the lens of HIV?

  40. #40 RRM
    September 5, 2011

    @genie

    I was not referring to the Lombardi et al. study, but to the study that David linked to:

    http://merutt.files.wordpress.com/2011/05/mikovits_lombardi-publikasjon-mai2001.pdf

    In this study, Mikovits and her co-authors did exactly what I said they did.

    By the way: now that the authors have replaced “CFS patients” by “XMRV positive CFS patients”, they have made this study (completely unnecessarily) dependent of the Lombardi et al. Science study. If Science would indeed decide to retract the XMRV study (no matter how you feel about this, it is not unlikely), nobody will be able to get funding to validate these immunological findings, just because the authors were stupid enough to link their results to another finding that is under heavy debate.

    @POL

    You should study grammarretroviruses.

  41. #41 POL
    September 5, 2011

    @RRM

    Can you answer these two simple questions.

    How do gammaretroviruses propagate?
    What is the natural reservoir of gammaretroviruses?

  42. #42 David
    September 5, 2011

    @RRM

    “just because the authors were stupid enough to link their results to another finding that is under heavy debate.”

    So how come no one on the In Vivo review board noticed this, or the intervening years of negative findings.

    Two years and these ‘scientists’ learned nothing, like fucking creationists.

  43. #43 Belfry
    September 5, 2011

    @ERV and RRM

    You agree that JohnCoffin’s paper, Paprotka et al, should be retracted by Science magazine for failing to mention the use of a third assay in the paper. An omission that misleads any scientists reading the paper to think that the PCR assay with no determined sensitivity was used to screen the later xenografts. That is a black and white reason to retract a paper. At no time is the use of an RT -PCR assay recorded in the published paper, but the data from that assay is in the paper.

    Vinay Pathak is on camera admitting this at CROI.
    http://app2.capitalreach.com/esp1204/servlet/tc?c=10164&cn=retro&s=20445&&dp=player.jsp&e=13725&mediaType=podiumVideo

    Paprotka should be retracted.

  44. #44 Jack
    September 5, 2011

    Hang in there Abbie ;)

  45. #45 RRM
    September 5, 2011

    @ David

    I am not arguing this a good reason for not accepting the paper (although it is really bizarre that the XMRV results were allowed to be presented without even the slightest notion of the negative studies). I assert that it’s just stupid for scientists to reframe their patient population from 100% secure to stand the test of time to less than 100% secure. (I’d say less than 1%, but your opinion may differ. Anyway, any step “back” is really stupid in my view, as the “XMRV” label doesn’t really add anything in this case.)

    @POL

    I am ashamed to tell you that I cannot answer any of these very simple questions. I do know however that they are completely irrelevant to the issue at hand.

    Lombardi et al. could (supposedly) reliably differentiate between CFS patients infected with a gammaretrovirus and persons that were not infected. Based on a SAMPLE OF BLOOD (or a derivative), without using any magical special collection protocol. The same lead scientists could again detect this gammaretrovirus, in the protected unblinded setting of their own lab, in the blood of 118 out of 118 (!!!) “Lake Tahoe” patients.

    Either their findings are true and reproducible or they are false. If these results are true, they should be EASILY reproducible given the reported reliability of the test using just patients’ blood.

    If they cannot reproduce their initial results, it is useless to suggest that perhaps the virus is propagating somewhere else, or that its natural reservoir is perhaps not in the blood. Yes, any known of unknown virus could be present in an unknown reservoir at or below the current limit of detection. However, that has nothing to do with the Lombardi et al. (and Lo) findings.

    @Belfry

    Sigh. No, it should not be retracted. You are misinterpreting the paper/conference talk.

    Do you realize that Pathak submitted the paper to Science before the CROI talk? Now, do you really think Pathak is so stupid that he would openly mention fatally damaging data in a publicly available talk that he had concealed earlier in the submitted manuscript?

    Nice video though:

    http://www.youtube.com/watch?v=mhW95ahBAgw&feature=player_embedded

    Seriously, that is one of the worst clips I have ever seen in my entire life. Compared to this, 2 girls 1 cup is Academy Award winning material. I dare anyone to watch that video from start to finish.

  46. #46 Benoni
    September 5, 2011

    HERP DERP!

  47. #47 Sox
    September 6, 2011

    @RRM

    Pathak does fatally admit it on camera in a talk about the paper. That is an automatic retraction.

    “In contrast if we do RT-PCR of RNA derived from these late xenografts we can readily detect XMRV.”

    Paprotka et al. has now embarrassed Science and the authors have been caught red handed.

    Last talk of the day.
    http://app2.capitalreach.com/esp1204/servlet/tc?c=10164&cn=retro&s=20445&&dp=player.jsp&e=13725&mediaType=podiumVideo

  48. #48 ERV
    September 6, 2011

    Pick one fucking screen name or Im banning the IP#, Mar/Shotman/Belfry/Sox.

  49. #49 RRM
    September 6, 2011

    Sox, I already know about that talk. In fact (and rather regrettably), it was only because I linked to this talk in another discussion, that this “fatal admission” got noticed. I suggest you take this very important finding up with Science (make sure to credit me) and I am sure they will take appropriate action.

    “We don’t do direct PCR.”
    Judy Mikovits 12/18/2010

    I’d say that looks more like an “automatic retraction”. ;-)

  50. #50 mary
    September 6, 2011

    belfry/Sox whoever…..

    that didn’t even get much reaction when you posted that on the: “omg I”m xmrv+, grand conspiracy, they are trying to kill us” forum…..

    OMFG that video RRM…sorry, couldn’t watch past the minute 50 mark..then my eyes started bleeding..

    don’t watch it unless you have insomnia and need to fall alseep…

  51. #51 Sox
    September 7, 2011

    @Mary

    Thank for the courtesy of not denying the omission from Paprokta and therefore being in agreement that the paper has to be retracted.

    “All data necessary to understand, assess, and extend the conclusions of the manuscript must be available to any reader of Science.” Science

  52. #52 POL
    September 7, 2011

    @RRM

    “Lombardi et al. could (supposedly) reliably differentiate between CFS patients infected with a gammaretrovirus and persons that were not infected. Based on a SAMPLE OF BLOOD (or a derivative), without using any magical special collection protocol. The same lead scientists could again detect this gammaretrovirus, in the protected unblinded setting of their own lab, in the blood of 118 out of 118 (!!!) “Lake Tahoe” patients.”

    The entire process has to be performed in a particular way. Any alteration from that does not challenge the results. The topic is virology not creationism. Lombardi et al (2010) is not Lombardi et al. (2009)

    “Either their findings are true and reproducible or they are false. If these results are true, they should be EASILY reproducible given the reported reliability of the test using just patients’ blood.”

    The word you were after is replication. Are they reproducible when replicated and that means you don’t change any parameter.

    “If they cannot reproduce their initial results, it is useless to suggest that perhaps the virus is propagating somewhere else, or that its natural reservoir is perhaps not in the blood. Yes, any known of unknown virus could be present in an unknown reservoir at or below the current limit of detection. However, that has nothing to do with the Lombardi et al. (and Lo) findings.”

    The virus is already known to not have its home in blood. If you don’t keep up with the research and read more than the abstract, these are typically ludicrous comments. Gammaretroviruses have never been found to ever do anything else. When working at the detection limits of PCR, it has everything to do with the findings in Lombardi and Lo. Replication is the only scientific option to refute the findings.

    @Jack
    “Hang in their Abbie.”

    The will make you believe you were not wrong, but there is no doubt gammaretroviruses are infecting people with ME and it will be back to haunt you.

  53. #53 Jack
    September 7, 2011

    I love the latest ‘evolution’ of the ‘XMRV’ theory, that now you can’t even find ‘it’ in blood and to look there – like as was done originally – is no longer ‘good enough’.

    Somehow or other the original Lombardi paper seems to have lost its’ significance even among the supporters. I mean if what happened in that original paper was ‘good enough’ to get those results, then WHY isn’t Mikerknickers doing exactly the same thing in the BWG and under Lipkin?

  54. #54 Jack
    September 7, 2011

    @POL

    ‘They will make you believe you were not wrong, but there is no doubt gammaretroviruses are infecting people with ME and it will be back to haunt you’

    Even if I have ‘it’ are you suggesting that ‘it’ causes and prolongs my symptoms?

    There is nothing definite about the understanding of my condition POL. Nothing.

    And when it comes to ‘XMRV’ and virology it is ALWAYS best to read as much as you can and gain an understanding of as much as you are able.

    Ask questions, never presume and try not to be led into thinking that there is one ‘answer’ that will end your pain.

    As I and others have said here and elsewhere many times before, I get the despair and I can appreciate – not understand – but appreciate what it might be like to be ‘tested’ and told you have an infectious retrovirus in your blood.

    I just can’t help but feel you and others are being led around by your despair and exploited and that this exploitation is now no different to what I and others with this diagnosis have experienced in the ‘alternative’ health markets and with other certified practitioners who have a ‘theory’ and are willing to ‘test’ it out on patients.

    There will never be an end to this for some who fail to consider the evidence placed before them. Pride comes before a fall – and that fall is long overdue for some scientists and pushers.

    As ever in science the door will never firmly be closed on ‘XMRV’ and so long as it isn’t then a few will continue to push against it.

    BWG and Lipkin will not be the end for them, and I am not predicting either study will be ‘negative’ either but even if it is – it will be argued (already is) that the studies were not adequate enough.

    I refer you to RRM’s quote above:
    “We don’t do direct PCR” Judy Mikovits 12/18/2010

    On certain forums and ‘advocate’ sites it is being claimed that the BWG are doing only Direct PCR on their samples – despite the authors and including Mikovits most recently denying this…

    When these studies are done – the claims will be that the evidence has been buried.

  55. #55 RRM
    September 7, 2011

    Exactly, Jack. What’s even more bizarre about this latest ‘evolution’: people like POL are arguing for ‘replication’ and ‘abandoning blood’ at the same time.

    “Nobody can change any parameter, but we really have to change this parameter.”

  56. #56 Jim
    September 7, 2011

    @RRM

    People are arguing for replication of the methods used in Lommbardi et al for blood. Methods that are diagnostically validated to work. The use of all methods in the prostate cancer positives to confirm the low level or even baseness in blood. And the use of tissue studies to find all infected people more easily and undercover the pathogenesis of HGRVs. It is a gammaretrovirus, it is not HIV. The blood is not their natural reservoir.

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