Trust science, not scientists

27 September 2011

XMRVWhether or not the retrovirus XMRV is a human pathogen has been debated since the virus was first described in 2006. The answer is now clear: the results of Blood XMRV Scientific Research Group, along with a partial retraction of the 2009 Science paper describing identification of the retrovirus in patients with chronic fatigue syndrome (CFS) show that detection of XMRV in patient samples is a result of contamination.

The Blood XMRV group obtained new blood samples from 15 individuals previously shown to be positive for XMRV (Lombardi et al., 2009) or MLV (Lo et al., 2010) ; 14 of these were from CFS patients. Fifteen blood samples were also obtained from healthy donors. The samples were coded and sent to 9 laboratories for analysis. These laboratories (Abbott Molecular, Abbott Diagnostics, CDC, FDA/Lo, FDA/Hewlett, Gen-Probe, NCI/DRP, and WPI) conducted validated assays for viral nucleic acid, viral replication, or viral antibodies. Positive control samples were also included that were ‘spiked’ with XMRV, in the form of cell culture fluids from the cell line 22Rv1. Each laboratory was at liberty to choose which assays to carry out.

Two laboratories reported evidence of XMRV in the coded samples.  Only WPI identified positive specimens by PCR: two from negative controls, and one from a CFS patient. The FDA/Lo laboratory did not detect any positives by PCR, using the same nested assay that they had previously reported in their published study. The samples tested included 5 specimens that were positive in the Lo et al. study.

Lombardi and colleagues have previously concluded that viral culture is the most sensitive method for detecting XMRV; however the FDA/Hewlett laboratory failed to culture virus from CFS samples. This laboratory did culture virus from positive control specimens, demonstrating the sensitivity of their methods. The FDA/Ruscetti laboratory recovered virus from 3/15 CFS samples but also from 6/15 negative control specimens. WPI did not carry out viral culture assays due to contamination of their cell lines with mycoplasma.

Four laboratories tested the samples for the presence of antibodies that react with XMRV proteins. Only WPI and NCI/Ruscetti detected reactive antibodies, both in CFS specimens and negative controls. There was no statistically significant difference in the rates of positivity between the positive and negative controls, nor in the identity of the positive samples between the two laboratories.

These results demonstrate that XMRV or antibodies to the virus are not present in clinical specimens. Detection of XMRV nucleic acid by WPI is likely a consequence of contamination. The positive serology reported by WPI and NCI/Ruscetti laboratories remained unexplained, but are most likely the result of the presence of cross-reactive epitopes. The authors of the study conclude that ‘routine blood screening for XMRV/P-MLV is not warranted at this time’.

One of the authors on Lombardi et al., Robert Silverman, decided to reexamine some of the DNA preparations from CFS patients that were originally used to detect XMRV DNA by PCR. He found that all the positive specimens from CFS patients were contaminated with XMRV plasmid DNA. Therefore the authors of the original study have retracted Figure 1 (single-round PCR detection of XMRV in CFS PBMC DNA); table S1, XMRV sequences, and figure S2, phylogenetic analysis of XMRV sequences.

A puzzling aspect of Silverman’s results is that XMRV plasmid DNA was detected only in samples from CFS patients, not healthy controls. This pattern would not be expected if the specimens were properly blinded, that is, coded so that the investigators did not know which were controls and which were from CFS patients. The authors offer no explanation of these findings.

The paper reporting contamination of samples with XMRV is entitled ‘Partial Retraction‘. It’s not clear to me why the entire paper has not been retracted. After removing the PCR and nucleic acid sequencing results, there is no evidence indicating the presence of XMRV in the patient samples. The remaining experiments show detection of a retrovirus by cell culture experiments, and the presence of viral proteins or antibodies to the virus in clinical specimens. None of these findings prove that what is being studied is XMRV. The title of the original paper ‘Detection of an infectious retrovirus’, XMRV, in blood cells of patients with chronic fatigue syndrome‘, is unsupported.

In an accompanying article on the XMRV story entitled ‘False Positive‘, Judy Mikovits of WPI notes that “Anyone who says this is a lab contaminant has drawn the wrong conclusion and has done a disservice to the public”. She goes on to imply that a gammaretrovirus is likely involved in CFS. On the contrary, pursuing the CFS-gammaretrovirus hypothesis is a disservice to those with CFS, and detracts from efforts to solve the disease. There are no data to support such an association, and to suggest that a lab contaminant, XMRV, has pointed the way to a bona fide etiologic agent seems implausible.

XMRV does not cause CFS. The virus arose in mice between 1993-96, and its detection in patient samples is clearly a result of contamination. Reaching these conclusions has required a long and often contentious journey that has highlighted the best and worst aspects of scientific research. There are many lessons to be learned from XMRV, but an important one is that science progresses not from the work of a single investigator, but from the collective efforts of many laboratories. XMRV reminds us to trust science, not scientists.

  • window

    Two quick examples, and then a partial defence of ‘science':

    re personality disorders:

    This is the biggest study to make claims of a link between CFS and personality disorders: http://content.karger.com/ProdukteDB/produkte.asp?Doi=319312

    It was promoted with a press release titled ‘Is Chronic Fatigue Syndrome a personality disorder?’, and got a lot of coverage.

    http://www.rehacare.com/cipp/md_rehacare/custom/pub/content,oid,27211/lang,2/ticket,g_u_e_s_t/mcat_id,7772/local_lang,2/~/Is_Chronic_Fatigue_Syndrome_A_Personality_Disorder.html

    If you read the paper, you can see that the answer is ‘no’.

    Those with CFS are more likely to fulfil the criteria for a personality disorder than the healthy population, but not more likely that the control group with health problems.  The paper acknowledged that previous studies comparing CFS and MS had also found the same levels of personality and psychiatric disorders.

    When personality disorders are diagnosed according to the individuals ability to fit in with society, it’s inevitable that those with chronic health problems will be more likely to fulfil the criteria, and when you look at the PDQ4 questionnaire used, this point is particularly clear.

    re PACE and CBT/GET – That the researchers started their study claiming that an SF36-PF score of 85 indicated recovery and a score of 65 indicated severe and disabling fatigue, yet by their press conference were claiming that patients with an SF36 PF score of just 60 should be considered back to normal, reveals rather more about the efficacy of the treatment they were promoting, and their honesty, than they might realise.

    We should not just trust scientific consensus, nor the abstracts of papers, and certainly not the claims researchers make in the press releases – particularly with CFS, but science does give us the tools and information needed to pick apart false claims.  With CFS, we’ve seen a lot of bad science, where the prejudices of researchers have been imposed upon data that does not support their conclusions, and not nearly enough interest from sceptics keen to pick apart the misleading claims made about patients, but the scientific process has still provided us with some meaningful data, and allowed us to see how misleading the claims made by some ‘experts’ are.

    So long as there is so little interest in criticising poorly done papers which increase to the stigma and burden faced by patients, there’s going to be an ongoing culture of distrust between patients and the medical community.  I had some hope that this might be starting to change, partially thanks to the XMRV story, but I’m not sure that it ever will.  Trusting ‘experts’ and authorities helps simplify life, and I’m not sure how interested many are in engaging with the complexities and uncertainty of a condition like CFS, and pushing to hold those who have treated patients poorly to account.

  • RRM

    I have a reliable source (see attachment). That is something you cannot change.

    And why then isn’t Mikovits doing anything about this injustice? Perhaps she is too busy with changing the legends of some the figures in the Science papers? Oh no, another controversy coming – are there any postdocs left to blame? ;-)

  • RRM

    Yet you say that they didn’t touch them.

    I now showed you that:
    1. NCI/Ruscetti tested them before the BWG’
    2. NCI/Ruscetti tested them in the BWG
    3. The test results do not match.

  • OWE

    I found this in the supplementary information of the original Lombardi paper:Quote
    “Figure S3. Detection of cloned XMRV-VP62 using a rat mAb to SFFV Env and agoat antiserum to mouse NZB xenotropic MLV. A. Lysates were prepared fromXMRV-VP62-infected Raji (lane1), LNCaP (lane 2) or Sup-T1 (lane 3). Positive
    Figure S3. Detection of cloned XMRV-VP62 using a rat mAb to SFFV Env and agoat antiserum to mouse NZB xenotropic MLV. A. Lysates were prepared fromXMRV-VP62-infected Raji (lane1), LNCaP (lane 2) or Sup-T1 (lane 3). Positive
    goat antiserum to mouse NZB xenotropic MLV. A. Lysates were prepared fromXMRV-VP62-infected Raji (lane1), LNCaP (lane 2) or Sup-T1 (lane 3). Positive
    XMRV-VP62-infected Raji (lane1), LNCaP (lane 2) or Sup-T1 (lane 3). Positive
    8controls used were HCD-57 cells, a mouse erythroleukemia cell line expressing
    polytropic MLV gp70 Env (lane 4), and HCD-57 cells infected with SFFV, which
    also express SFFV gp55 Env (lane 5). WB analysis was carried out using rat
    anti-SFFV Env mAb 7C10. Molecular weight markers in kD are shown on the
    left. B. Lysates were prepared from XMRV-VP62-infected Raji (lane 1), LNCaP(lane 2) or Sup-T1 (lane 3). Lysates from SFFV-infected mouse HCD-57 cells(lane 4) and from uninfected Raji, LNCaP and Sup-T1 are shown in lanes 5-7,respectively. WB was carried out using goat antiserum to mouse NZB xenotropicMLV. Molecular weight markers in kD are shown on the left.”This shows that they had VP62 in their labs. OWEB. Lysates were prepared from XMRV-VP62-infected Raji (lane 1), LNCaP(lane 2) or Sup-T1 (lane 3). Lysates from SFFV-infected mouse HCD-57 cells
    (lane 4) and from uninfected Raji, LNCaP and Sup-T1 are shown in lanes 5-7,
    respectively. WB was carried out using goat antiserum to mouse NZB xenotropic
    MLV. Molecular weight markers in kD are shown on the left.”

    This shows that they had VP62 in their labs.
    OWE

  • Anonymous

    That is not a source.  Cohen is not a scientist.  And what don’t you know, the VP62 plasmid was never in the WPI or NCI labs.  Are you again questioning the integrity of Frank Ruscetti, Mikovits and Silverman by suggesting they would always go out of their way to correct falsehoods like yours and if they didn’t you would want people to think they lied?  They haven’t done that with several studies either, so who do you think you are to question their honesty.   

  • RRM

    I gave you contact info and Frank has apparently refused to contact me.

    And that is a source that is considered to be reliable for the purpose of this discussion:
    http://en.wikipedia.org/wiki/Journalism_sourcing

  • Anonymous

    Looks like your comments have made you untrustworthy.  The VP62 plasmid was never in the WPI or NCI labs.

  • Anonymous

    That is Silvermans assay, performed in Silvermans lab, that you are incorrectly quoting. The VP62 plasmid was not in the WPI or NCI labs.

    The WPI used VP35 for Nested RT-PCR.

  • Anonymous

    Fractions of patient samples who had tested positive for gag sequences by reverse transcriptase nested PCR were exported to the Cleveland clinic for sequencing.  When the retained fractions were independently examined they did not contain the VP-62 plasmid. Following single round PCR at the Cleveland clinic the exported fractions were found to contain the VP-62 plasmid.  Dr Silverman made an honest mistake and was brave enough to reveal it.

  • Anonymous

    You think that Busch is going to be happy that he didn’t add Trizol to the PBMCs so that the WPIs assay was defeated due to the viruses being degraded.  I’m sure he doesn’t like you making light of it. 

  • OWE

    Are you contradicting Judy Mikovits? The section is taken from the Supporting Online Material that was published together with the original Lombardi paper in 2009.  Have a look at it yourself, you will find it on pages 7/8 in the “Supporting Online Materials and Methods” section! It demonstrates clearly that XMRV-VP62 was handled at the WPI labs for some experiments and Judy Mikovits surely knows what she is publishing… 

    Where is your proof that WPI never had VP62 plasmid? Are you working at the WPI to be able to say that?

    OWE  

  • OWE

    Are you contradicting Judy Mikovits? The section is taken from the Supporting Online Material that was published together with the original Lombardi paper in 2009.  Have a look at it yourself, you will find it on pages 7/8 in the “Supporting Online Materials and Methods” section! It demonstrates clearly that XMRV-VP62 was handled at the WPI labs for some experiments and Judy Mikovits surely knows what she is publishing… 

    Where is your proof that WPI never had VP62 plasmid? Are you working at the WPI to be able to say that?

    OWE  

  • Anonymous

    The VP62 plasmid was never in the WPI or NCI, that is not hard to understand, even if you post the above without understanding it.

    You should understand this.  Fractions of patient samples who had tested positive for gag sequences by reverse transcriptase nested PCR were exported to the Cleveland clinic for sequencing.  When the retained fractions were independently examined they did not contain the VP-62 plasmid. Following single round PCR at the Cleveland clinic the exported fractions were found to contain the VP-62 plasmid.  Dr Silverman made an honest mistake and was brave enough to reveal it.

  • Anonymous

    RRM studies like this would not allow work that took place outside of the study, which was to see if there was a blood test that could be high throughput.  There isn’t.   They have nothing that will protect the blood supply and find all HGRVs.

    The WPI and NCI never tested the Lo samples before blinding for this study

  • OWE

    Where is your proof that WPI never had VP62, when theypublish exactly the contrary in the supporting online material to their 2009 Lombarid et al. paper (did you actually read it?).

    OWE

  • OWE

    Have you seen the latest post by ERV?

    XMRV and chronic fatigue syndrome: For your enjoyment– A magic trick.

    Very interesting for all those that believe in Judy’s honesty…

    OWE

  • http://www.facebook.com/people/Heather-Martin/100002322342051 Heather Martin

    Interesting that while the opinion over whether contamination occurred or not rages on… no one is discussing, why the CFS patients have CFS.  XMRV may or may not be the culprit, and this article merely begs that follow up occur to settle the matter.  As well, would it not be prudent to look at other culprits such as  aluminum based adjuvants.  Imagine if those were the cause of the patients CFS?  It makes the debate over 
    XMRV kind of a moot point.  I say repeat the experiment, compare the new results with the old and then move on.

  • drosha

    Why do you keep writing this? Trizol is a preservative agent for RNA, not viruses. Retroviruses don’t require Trizol preservation. This claim shows that you have no idea what you’re talking about, and have no research experience.

  • Jack

    ‘We’re going around in circles here. We never stated that VP62 contaminated
    samples at WPI. Whether WPI had VP62 is a separate question, and, as I
    have detailed, both Mikovits and Silverman say the institute had it. I’m
    uncertain why you continue to insist otherwise, and this is my last
    reply on this question.
     
     Note, too, that in the partial retraction, supporting online material, it states in reference to Silverman’s group at Cleveland Clinic: “In particular, neither plasmid XMRV VP62/pcDNA3.1(-) nor XMRV PCR products were ever taken into the clean room.” I also refer you to figure 3 in the original supplementary material for Lombardi et al., which adds more details about the use of VP62 for serology tests.’http://news.sciencemag.org/scienceinsider/2011/09/insider-looking-out-how-people.html#sci-commentsEarlier today. Unless Jon Cohen was replying to you, you might not have seen this.

  • Jack

    ‘You and other can
    keep repeating that VP62 was never in the WPI labs, but it does not
    change the facts, as described to me in detail by both Judy Mikovits and
    Robert Silverman.
     

     
    I think the confusion may stem from a May 30 letter that Judy Mikovits sent to Bruce Alberts in which she writes:
     

     
    “Neither 22Rv1 nor any of the cell lines reported to be contaminated with XMRV or cell lines growing the VP62 infectious molecularly cloned virus was in the laboratories where the patient cells were isolated.”
     

     
    (Judy Mikovits, by the way, not Science, gave me a copy of this letter.)
     

     
    Note that this does not say anything about whether VP62 was at WPI,
    which Silverman told me he sent to WPI in March 2008. Judy Mikovits
    said to me, “We had VP62 as a clone.”
     

     
    Whether VP62 at WPI could have contaminated patient samples is another question. But I have told you my sources for the statement I made. Who are your sources?’

    ‘We’re going around in circles here. We never stated that VP62 contaminated
    samples at WPI. Whether WPI had VP62 is a separate question, and, as I
    have detailed, both Mikovits and Silverman say the institute had it. I’m
    uncertain why you continue to insist otherwise, and this is my last
    reply on this question.

     
    Note, too, that in the partial retraction, supporting online material, it states in reference to Silverman’s group at Cleveland Clinic: “In particular, neither plasmid XMRV VP62/pcDNA3.1(-) nor XMRV PCR products were ever taken into the clean room.” I also refer you to figure 3 in the original supplementary material for Lombardi et al., which adds more details about the use of VP62 for serology tests.’

    http://news.sciencemag.org/scienceinsider/2011/09/insider-looking-out-how-people.html#sci-comments

  • RRM

    prop.logic gap in post http://www.virology.ws/2011/09/29/admit-when-you-are-wrong/#comment-324195705

    Check if someone spots it

  • Guest

    All of the sequences the Whittemore Peterson Institute uploaded to Genbank were identical.They were not only identical to one another, they were identical to an infectious molecular clone of XMRV, VP62.
    There is no research data or paper to date that proves HGRV causes ME. To state otherwise is sheer lunancy!

    Silverman sent the WPI VP62 plasmids in 2007 (before they ‘found’ XMRV in patient samples.
    The CFS samples that left WPI were contaminated with VP62 pasmid prior to Silvermann lab receiving them in 2009. Lombardi paper states all of these ABs detected the human VP62 XMRV strain grown in human Raji, LNCaP, Sup-T1 cells (fig. S3)(5).
    Silvermans lab found VP62 plasmid in their samples. Which means the positives Silvermans lab reported in 2009 were all because of plasmid contamination. The positive results were NOT the result of real infection.

    Silverman only found VP62 plasmid in the samples he got from the WPI… and only in the CFS patient samples. Not the healthy controls

  • Richard Jefferys

    Darkow wrote:

    “HIV was also incorrectly called HTLV-II for a while and was not cloned for at least 10 years after its discovery.”

    You’ve tried that lie before. Under a different username. 

    http://www.ncbi.nlm.nih.gov/pubmed/6096717

    Nature. 1984 Dec 20-1985 Jan 2;312(5996):757-60.
    Molecular cloning of lymphadenopathy-associated virus.
    Alizon M, Sonigo P, Barré-Sinoussi F, Chermann JC, Tiollais P, Montagnier L, Wain-Hobson S. 

    – Gallo’s original name for HIV was HTLV-III, he held out for as long as possible before adopting the new name (http://www.nytimes.com/1986/05/01/us/new-name-is-proposed-for-the-cause-of-aids.html)

  • Sciencewatcher

    OWE, the WPI and NCI, even thought you refuse to accept this, never have had VP62 plasmid in their labs.

  • Sciencewatcher

    The VP62 plasmid has never been in the WPI or NCI labs.

  • Sciencewatcher

    Worth repeating.

    ” Without trizol the PBMCs degrade and so does RNA. You need to demonstrate a specific antibody response which only occurs if a gammaretrovirus of a certain class is present and the presence of viral proteins. The WPI assay does not look at proviruses but at the viral RNA, hence the degredation of PMBCs and the degredation of RNA would have defeated the test. The PBMCs were not trizoled post extraction which would have rendered them useless. Apparently you fail to understand such a simple point.”

  • Sciencewatcher

    Several varieties of retroviruses do have highly conserved regions.  They don’t use reverse transcriptase to propagate and thus there diversity is small.  HTLV has a 1% change over 1000 years in the whole genome.  

    The samples that Silverman had were contaminated with the VP62 plasmid, the samples when sent were not contaminated with the VP62 plasmid, and the VP62 plasmid has never been in the WPI or NCI labs.  Silverman never sent the WPI or NCI the VP62 plasmid. 

  • Mtmail

    RRM, you are a true soldier, thanks for taking the time to discuss with crazy people.
    keep up fighting propaganda for all of us!!

  • http://profiles.google.com/judderwocky Judson Wallace

    They are not necessarily questioning their integrity…. any claim like that would have to be verified…

    I mean, really he’s making the same argument that CFS patients were originally arguing…. a single lab cannot be counted on to accurately test for a negative like that….

    So really its only fair to bring it back the other way around: Mikovits cannot by herself show that her samples are free of those viral contaminants…

  • http://profiles.google.com/judderwocky Judson Wallace

    Sucking money up for research that is clearly debunked is a disservice to those CFS patients.

    There are probably BILLIONS of different viruses in the world…. focusing on one  bad lead IS A DISSERVICE

    Take a step back and remember that the entire team at virology.ws pushed this research when few else would.

    It has been given a fair run, but it does not appear that XMRV/HGRV/HGTV is the culprit.

  • Sciencewatcher

    Yes, you can see that the last images on there have been shifted.

  • drosha

    It’s worth understanding what Trizol is first:

    Trizol is a monophasic solution of phenol and guanidine isothiocyanate. It’s used mostly in RNA isolations from cells or other tissue samples mostly because phenol is a potent disruptor of cell membranes and guanidine isothiocyanate is a protein denaturant. If you add this solution to a virus prep or cells, you cannot use those specimens for further culturing. Sure, you’ll be preserving the RNA in these specimens but most of the proteins would be denatured and cell membranes would be disrupted leading to destruction of the cells and the virus. Hence, Cells don’t degrade without Trizol, they degrade with Trizol. If your sole purpose is to isolate RNA from cells then Trizol would be a great addition to your specimens for RNA preservation, but if you’re planning on doing other experiments, it’s a bad idea to use Trizol.

    Obviously if you don’t know what Trizol is (or understand what its ingredients do) you’d be like the above poster; keep repeating something you heard from an uninformed person.

  • drosha

    “They don’t use reverse transcriptase to propagate and thus there diversity is small.”

    This definitely convinced me that you have no idea what you’re talking about.

    There is no retrovirus (or a region of retrovirus) that doesn’t use reverse transcriptase.

  • Justin Reilly, esq.

    How about if they changed “XMRV” to “HGRVs” in the title of the paper instead of retracting the paper?  The data support that title, right?

    Dr. M says she will reveal the sequences of the HGRVs she found in about a week.  So then we can just substitute those sequences in for “XMRV.”Cleveland Clinic’s understandable mistake doesn’t impugn the rest of the paper, Ruscetti or WPI.  So why all the attack on Lombardi et al and WPI because of the partial retraction?

  • Lance

    I would REALLY like to see you prevent PBMCs from “degrading” using TRIzol. I think you’ll find it kills them instantly! Just get hold of some and give it a (cautious) sniff….

  • Lance

    You can’t demonstrate and antibody response out of a sample preserved in TRIzol…. is that what you were implying?

  • Lance

    Sorry…

    You can’t demonstrate AN antibody response out of a sample preserved in TRIzol…. is that what you were implying?

  • Tony Mach

    Interesting. Nothing to back up the claim, with contrary sources. And still it comes up. Again, and again, and again. Like a broken record.

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  • linzel

    I’m afraid no one does trust either any more. We need to trust science, trust scientistS and be skeptical of the lone ranger scientist. The lack of trust in the first two cause ‘denial’ when the scientific debate is nearly complete or better. Evolution, climate change, ozone hole, second hand smoke, acid rain are but some examples of how collectively scientistS CAn and need to be trusted. As a group, not as individuals. Perhaps this is one aspect of trusting science. I’m afraid the pendulum of trusting experts in a a field is at an all time low. I think this destructive.

  • linzel

    That was a horrible first sentence, sorry. I’m too scared to reread the rest. Hope you understand what I meant. 

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