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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. currer

    currer Senior Member

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    I was at the IIME conference yesterday and Dr Peterson gave a rapid talk in which he updated us on all the ME studies that are ongoing in the US at the moment.
    He started by describing a study looking for XMRV, although he did not introduce it as "the Lipkin study" so I only realised this must be what he was refering to after he had moved on to the next topic.
    It seemed quite a small study, the patients and controls carefully matched, and the samples were spread among three labs, one of which was named "the Mikovits lab in New York".
    This study is completed and the results will be released on the 30th June, and he said it "should draw a line under XMRV" and from the tone of his voice it sounds as if he knows the result to be negative.
    He then moved on to describe another study looking for pathogens, and this was when Lipkin's name was mentioned for the first time, and I realised that the first study must be what we refer to as the Lipkin study.
    I cannot be certain of the exact details as I was too unwell to take notes at the time. I initially thought I would be too ill to go, and only changed my mind at the last minute and had to miss the first part of the conference as a result.
    Xandoff and Bob like this.
  2. Xandoff

    Xandoff Michael

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    Currer..........Thank you for this posting and your courage to be there. I guess we will be waiting on the American Healthcare Act to pass by the Supreme Court and for these studies to break.
  3. acer2000

    acer2000 Senior Member

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    In my mind one of the biggest questions raised by the 2009 Lombardi paper and subsequent studies that produced similar results is the fact that in the studies that actually could detect XMRV/MLVs the patient population had many more positives than the control population. I actually think its quite interesting the amount general information that has come out about the history of XMRV and its phylo-genetic roots as well as how easy it is to contaminate labs and cell lines with similar viruses. But at the end of the day, someone needs to explain why, when controlling for all of these things, sick people show up positive on these tests more than controls. I really hope that if the Lipkin study is in fact "negative" they answer this question definitively. Otherwise it really won't be all that helpful of a study.
    beaker, natasa778 and jace like this.
  4. barbc56

    barbc56 Senior Member

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    Natasha, this is not true. The controls in the original study with Mikovitz et. al. were not handled the same . Here's something that is worth repeating:

    http://www.research1st.com/2011/10/14/xmrv-updates/

    I think this study was pretty definitive but hope the Lipkin study will be the final word. Positive or negative!!

    I know there are some in the community who don't think this study was valid. IMHO, it was.

    Barb C.


    One thing as far as detetcting MLV fragments (?), etc., the more sophisticated the detection level, the more likely you are to pick up background noise. I will try to get more information about this though I think many scientist have said the same thing.

    Also the fact that the patient blood was spiked. I can't remember if this was known at the time the above was written.
  5. barbc56

    barbc56 Senior Member

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    Here it is in full:

    From the webinar:Answers to 10 Common Criticisms of the SRWG study
    by Graham Simmons, PhD

    1. All of the controls were not screened by all of the labs.
    Response: Controls were screened by at least five labs: WPI, National Cancer Insitute/NCI-Ruscetti, Food and Drug Administration/FDA-Lo, Centers for Disease Control & Prevention (CDC) and NCI/Drug Resistance Program (DRP).

    2. Control peripheral blood mononuclear cells (PBMCs) were not screened prior to blinding, so could not have been ruled as negative.
    Response: Three out of the 15 did have their PBMCs extensively screened prior to blinding, yet two of these were still called “positive” in various assays by the WPI and NCI/Ruscetti in the study.

    3. No cryopreservative was used for the storage of the PBMCs, which would prevent the WPI’s assay from working. No Trizol was used.
    Response: Due to the short-term nature of the study it was not felt that preservatives were required for PBMC cryopreservation. The Lo/Alter study detected sequences in PBMCs stored for 15 years in the absence of preservatives. Trizol is for the extraction of nucleic acid and laboratories were given the option of choosing their own extraction methods

    4. The length of time allotted for the serology and culture assays was massively reduced, so that the WPI or NCI/Ruscetti assays were not performed as desired.
    Response: All the laboratories were allowed as much time as required to perform their desired assays. The culture and serological assays were performed by WPI and NCI/Ruscetti to their own specifications.

    5. The WPI was not given the opportunity to complete virus culture assays.
    Response: The WPI encountered mycoplasma contamination of their target cell population, and used the plasma samples without results. This was very unfortunate. There were no further stocks left to perform repeat cultures with. It was deemed by both the WPI and the working group that performing the studies on freeze/thawed material would be invalid.

    6. Samples and collection tubes were handled in the same laboratory as 22Rv1 cells used to spike the analytical controls.
    Response: As stated in the paper, 22Rv1 cells were handled in a separate facility to where all other activities were performed. The fact that only one laboratory detected PCR and virus culture in clinical samples supports the fact that 22Rv1 contamination did not occur at the central laboratory.

    7. Patients were on additional therapies that would produce false negatives.
    Response: Lo/Alter patients were not on any additional treatments. It is unclear what additional treatments patients were on at the time of Lombardi et al. There is no published evidence that additional treatments would have positive or negative effects.

    8. FDA/Lo used the wrong assay from Lo et al. and instead used the one that could not detect positives.

    Response: Lo et al. used their own criteria to decide on which assay(s) to use, but it is clear that both primer sets in their paper are equally capable of amplifying diverse polytropic murine leukemia viruses (MLVs), so it is not obvious that one would be better that the other at detecting “positives.”

    9. The NCI-Ruscetti did no PCR and could not use their clinically validated serology and culture assays.
    Response: NCI-Ruscetti felt that they were not sufficiently experienced at PCR to participate in the study. They did perform their serology and culture assays – just as performed in Lombardi et al.

    10. All the SRWG labs optimized their assays to VP62. VP62 does not exist in nature and Lombardi et al. is now known to have discovered HGRVs. Does your study include HGRVs? Or how do HGRVs relate to XMRV?

    Response: As demonstrated in an earlier slide, although this study was initiated after Lombardi et al. as a study of XMRV, as soon as Lo et al. was published the mission of the study was broadened to include all MLV-like viruses. Thus, almost all of the assays were designed to perform against MLVs in general and were optimized and tested as such. As our study has demonstrated there is no such thing as an independently validated clinically positive sample against which to test. Currently there is no such thing as human gammaretroviruses (HGRV). No published virus has been isolated, cloned or sequenced from a human.
  6. natasa778

    natasa778 Senior Member

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    What is not true?
  7. jace

    jace Off the fence

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    "Blood specimens were collected multiple times from three consenting laboratory controls under local IRB approval and WB, plasma and PBMCs were tested by all of the participating laboratories for XMRV/MLVs, using nucleic acid amplification testing (NAT), serology and virus culture techniques." - SOM

    There were 3 lab techs that were used as controls. Only those 3 were screened by all labs, using all methods. A lab tech working with the viruses is likely to become infected. So they could have been negative before blinding and positive after. The other controls were not screened with all methods by all labs and the blood group are not saying if any clinically validated method was used on any of the controls.

    "Similarly, fifteen control specimens from blood donors (n=12) or laboratory controls (n=3) that had been established as negative for XMRV and MLVs by PCR, serology and culture by multiple laboratories, were collected, processed and aliquoted in parallel (17)." - Main text
    The 'or' indicates they could choose either the 3 lab controls or the 12 blood donors to send to those labs. Note, they screened for XMRV and MLVs. Not MRVs, more on this at the end of this post.

    The 3 were the lab techs, not the people who were used as controls. Lab techs are exposed to the virus. They should not have been used as controls. The other 12 did not have their PBMCs screened prior to blinding.

    The study should not have been short term. They should be protecting the blood supply, and it doesn't matter how long it takes, only that it is done correctly. No trizol and no preservative was used, which stopped the WPIs assay from working.

    Because the PBMCs were not preserved in trizol and also exposed to a slow thaw, the samples would have been useless as far as the sophisticated techniques of Dr Ruscetti were concerned. Dr Lombardi being inexperienced simply did not notice. Matrix effects induced by poor sample prep affect the avidity of antibodies and hence the antigen antibody bonding will not survive the various washing processes.

    This changed the assays from being clinically validated to not being clinically validated. They rushed the study. The study has to be done again, correctly this time with only clinically validated assays.

    Again they did not design the study well enough and should have had these available, if they wanted to get an accurate result. This is the blood supply, not a science fair. The study should be redone correctly!

    22Rv1 was in the CDC lab with a portion of collection tubes, so yes one labs samples could be contaminated.
    "Spiked controls
    22Rv1 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. In order to create positive controls for NAT assays for PBMC and WB, ten 22Rv1 cells per aliquot of PBMC and WB from one of the three laboratory controls were added.
    For spiking of plasma, a large batch of supernatant from 22Rv1 cells was prepared from a T-75 flask plated at 60% confluency and grown for four days. Supernatant was collected and 0.45 μm filtered. An aliquot was tested by the CDC laboratory using six different real-time RT-PCR assays (19, 24-26). " - SOM

    "A single lot of EDTA BD Vacutainer Blood collection tubes (BD Biosciences) was purchased and distributed to two laboratories (CDC and NCI/DRP) for testing to ensure contamination with XMRV or mouse DNA was not present." - SOM

    The WPI patients were on additional therapies. We don't have the information about the others. There are loads of studies that show these treatments would affect results! Many drugs have an anti-retroviral effect, from Doxycycline on. Hands up who's not got a pill-sorter nearby?

    It doesn't matter that they chose it. They still chose to use an assay that had never found a patient positive... Primers are only one variable that can be altered to change the sensitivity of an assay. There is buffer, salts, magnesium, annealing temperature...

    So did the NCI tell Ruscetti he could not run PCR? That reply suggests they did.
    The culture and serology found positives. The issue is the messing up of controls previously. There were no true controls in the study. Those people are positive for the viruses!

    An assay designed to perform against MLVs is an assay designed to detect mouse viruses, not MRVs. The retroviruses found in ME patients are MRV's. The Genbank bank WPI env show this. They could not have broaded the assays. All PCR assays were optimised to a synthetic virus (VP62), not a wild type virus, or any other virus. See Hanson's study. She used a clinical positive to validate.

    Lombardi, who performed testing for this study, changed the PCR assay and it was not the assay from Lombardi et al. The assay he used here is not clinically validated.
    Lombardi et al. isolated the viruses. Others have isolated too, cloned and sequenced. VP42 and VP35 are clones of the prostate cancer HGRVs. They are not VP62. VP35 was cloned before VP62.


    I have a whole selection of quotes where countries admit to HGRVs. Let me know if you'd like to see them.

    Bell's patients in the Hanson study were the MRV positives. There was no positive in Levine's patients. Bell uses the CCC, Levine the Fukuda. What diagnostic protocol did the BWG III use? Apart from the Lo patients, it was Fukuda, which diagnoses +/-40% wrongly. Alter and Lo seem to have used Fukuda too, though it is not explicitly spelled out in the paper as far as I can see. Certainly the only diagnostic reference is to Fukuda.

    And then we come onto the question of coding - who, how, where, when? And that's another nest of worms, for BWG III and for Lipkin.

    We know very little about the Lipkin study. If it is a replication of Lombardi, even then it cannot be called definitive. We already know that the doctors providing the patients used Fukuda, which means it's not a replication, as Lombardi et al used the Canadian. I do hope the full paper is open access...

    You cannot have a definitive study. That is politics, not science. Science is a process of seeking to get as close to the truth as possible, but one cannot ever say "It is this way, and there is no other way" as our understanding of the natural world is continually evolving. Unless, of course, you want to shut the door on progress...
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  8. barbc56

    barbc56 Senior Member

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    Don't need to see the citations. I have heard/read these same arguments over and over and they have been refuted over and over by credible scientist. It's a mantra by a few people who claim to represent our community. A minority who appear to think they know the science and spin it according to their preconciived notions.

    We need to move on. Unless of course you want to shut the door on progress.;)

    IMHO!!

    Barb C.:>)
  9. jace

    jace Off the fence

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    Where? Who?
  10. Bob

    Bob

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    "We need to move on"? Do you mean that we need to move on from XMRV?
    I trust you are speaking for yourself Barb, and not stating what you think others should be doing with their lives?
    When we get the Lipkin results then each of us will decide for ourselves where our interests in the subject will go from there.
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  11. floydguy

    floydguy Senior Member

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    Dr. Lipkin disagreed with you by taking on the study with Dr. Mikovits et al. It's curious that we have less than a month for his report and people make such statements as above. Please clarify as to who the "credible" scientists are that you are referring to. I'll take Lipkin over Stoye/Coffin or your assessment of the situation any day of the week. I have yet to see you make any compelling science based arguments, only scary authoritarian declarations that progress can't be made if one is interested in what Lipkin has to say about XMRV.
    RustyJ and jace like this.
  12. jace

    jace Off the fence

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    "All samples were blinded, as mandated by the NCI and WPI institutional review board approvals. All experimental procedures were done by the same personnel, in the same physical laboratory space, under identical protocols. Investigators at NCI received 100 samples from individuals without knowing their health status; furthermore, the samples were sent to NCI directly without passing through the WPI laboratory space. Laboratory workers at the NCI and the WPI who performed the polymerase chain reaction (PCR) and immunological studies used coded, blinded samples that did not reveal the CFS status of the individuals. The WPI has examined all 218 control and 101 patient samples by both PCR and serological methods for the presence of XMRV nucleic acid and antibodies."
    http://www.sciencemag.org/content/328/5980/825.4.full.pdf
  13. anciendaze

    anciendaze Senior Member

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    I've stayed out of this scrum, but have been watching. I liked the comment by Mark on the lopsided results. I started trying random contamination models back in 2010, and failed to make them work on any results, including Huber's. Systematic contamination was the only possibility, but this has simply not been tied to patient samples.

    My reading of the Hanson/Bell results matches Sam Carter's:
    The state of the art for detecting contamination has reached such a level you can find it just about anywhere.

    The question which has apparently never been addressed is how anyone could use present techniques to find a retrovirus which might be causing a disease like this, or MS, RA, lupus, etc. If there were such a virus it would have to have extremely slow replication to persist without killing the patient in a year or three.

    Virologists seem to be telling us "we don't do chronic disease", except HIV. They seem quite comfortable for example with the morass around research on viruses in breast cancer. We can explain 95% of mammary tumors in mice, and what percentage in humans? How will doing the same thing, only more so, ever resolve this impasse?
    JT1024 and jace like this.
  14. Marco

    Marco Old blackguard

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    Just a thought but does anyone know what the respective treatments protocols are for the patients of these Drs?
  15. ukxmrv

    ukxmrv Senior Member

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    Barb,

    You have yet to produce any evidence that there are a "minority of patients who appear to think that they know the science and spin in according to their preconceived notions" so yes it is again your humble opinion.

    The same arguments you use could equaly be said of yourself and the researchers, journalists and commentators on MLV's that you quote.

    It may be that different parts of the "ME community" want different things. Quite possibly there is a different opinion on the direction needed from the older acute epidemic survivors, people with family members involved, with the opinions of the patients with a non-acute sporadic form of the disease.

    it opens that very old question - can the original ME survivors work with the people with CFS?

    It doesn't surprise me that different groups want different things investigated. There is no reason why money shouldn't be spend on the different areas though.

    I'm quite capable of following all the strands of ME research. There is a lot happening now from different areas and I'm happy with that.

    Not sure how you can argue that by following retroviral reseach into ME, which I have done since the first reports in the 80's, one could be shutting the door on progress?

    Why would you say that we would shut the door on progress? The question may boil down to which group's progress? Your progress may be different to others. Who exactly is your "we"?
  16. beaker

    beaker CFS/ME 1986

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    My emphasis(bold) in above quote.
    Thing is, I think we can all agree that what the whole community wants is to be better.
    If you keep that in mind, why oh why would any one want to shut down any viable research into the biopathology of this plague? Not sure "biopathology" is the right word but that's all my brain could come up with ;)
    and I needed a why to leave out the whole wesselys of the world.

    I do believe the truth of it will come out. I've been waiting a long time. (25+) I still wait. and hope.
    I hope they continue to explore this retroviral research. I hope they look at the autoimmune research. I hope they come up with new ideas. I don't want to wait another 25 to be better.
  17. Sam Carter

    Sam Carter Guest

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  18. barbc56

    barbc56 Senior Member

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    Hi, ukxmrv. This is an interesting statement. Would you expand on what this means? Has there been friction between those who come down with ME/CFS from an epidemic and other patients?

    Thanks.

    BarbC.:>)
  19. Bob

    Bob

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  20. barbc56

    barbc56 Senior Member

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    I tried but had some trouble getting it to work. I've done it before so will try again.

    Barb C.:>)

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