Negative XMRV study using PCR technology: PLoSone, 9 March 2011

March 10, 2011


From PloSone, an open access journal published by the Public Library of Science, 9 March 2011

Investigation into the Presence of and Serological Response to XMRV in CFS Patients

[ME Association quote from abstract: “Here, we revisit our patient cohort to investigate the XMRV status in those patients by means of the original PCR protocol which linked the virus to CFS.”]

Otto Erlwein1, Mark J. Robinson1, Steve Kaye1, Gillian Wills1, Shozo Izui2, Simon Wessely3, Jonathan Weber1, Anthony Cleare3, David Collier4, Myra O. McClure1*

1 Jefferiss Research Trust Laboratories, Section of Infectious Diseases, Wright-Fleming Institute, Faculty of Medicine, Imperial College London, London, United Kingdom, 2 Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland, 3 Department of Psychological Medicine, Institute of Psychiatry, King's College London, Camberwell, London, United Kingdom, 4 Social Genetic and Developmental Psychiatry Centre, Institute of Psychiatry, King's College London, London, United Kingdom

Abstract

The novel human gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV), originally described in prostate cancer, has also been implicated in chronic fatigue syndrome (CFS). When later reports failed to confirm the link to CFS, they were often criticised for not using the conditions described in the original study. Here, we revisit our patient cohort to investigate the XMRV status in those patients by means of the original PCR protocol which linked the virus to CFS. In addition, sera from our CFS patients were assayed for the presence of xenotropic virus envelope protein, as well as a serological response to it. The results further strengthen our contention that there is no evidence for an association of XMRV with CFS, at least in the UK.

Citation: Erlwein O, Robinson MJ, Kaye S, Wills G, Izui S, et al. (2011) Investigation into the Presence of and Serological Response to XMRV in CFS Patients. PLoS ONE 6(3): e17592. doi:10.1371/journal.pone.0017592

Editor: Kim Hasenkrug, National Institute of Allergy and Infectious Diseases, United States of America

Received: November 26, 2010; Accepted: January 26, 2011; Published: March 9, 2011

Copyright: © 2011 Erlwein et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the NIHR Biomedical Research Centre. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: m.mcclure@imperial.ac.uk

15 thoughts on “Negative XMRV study using PCR technology: PLoSone, 9 March 2011”

  1. This study used the same patients as the previous Erwin et al. (2010)

    They used PCR not Nested or RT-PCR. There was also no culture. The also used the VP62 clone to calibrate their assay to, as did every other negative study, thereby limiting what they would be capable of detecting. The ELISA assay was the one used by Hohn which didn’t work then, and in fact no ELISA has ever worked on this virus.

  2. ‘The connection between XMRV and CFS remains highly controversial. The initial report by Lombardi et al [10] identified XMRV in 67% of CFS patients and 3.7% of health control subjects. Subsequent to this, using the same experimental protocol an independent study by Lo et al. [11] detected four classes of MLV-related gag sequences in 86.5% of CFS patients and blood donors.

    In this study [11] it was suggested that MLV-related sequences found in CFS cases vary to the extent that they may have remained undetected by our original PCR primers which targeted the leader gag region and the pol region of XMRV. We have now used the primers described in the original paper [10] which bind to the XMRV gag and env open reading frames.

    We have again failed to detect XMRV or MLV-related sequences in 48 of our CFS patients, demonstrating that our failure to find XMRV in CFS tissue is not a reflection of the primers used in the amplification process.

    Indeed, the original primer sets have been exploited in several studies which have failed to amplify XMRV sequences in CFS patient samples [14], [16], [17].

    ‘However, this study confirms our previous report that a connection of XMRV or MLV-related viruses with UK CFS patients could not be substantiated.’

    Well that and the other stuff I could understand sounds pretty conclusive – no?

    I dare say this one will continue to rage on and on and on, until the original authors and especially the WPI eventually are able to do something about it.

    Personally, I am feeling rather numb now from the neck up.

    1. Firestorm,
      From the above comment, it would appear that you are one of the authors of this study and you should therefore declare who you are.

      1. Don’t be stupid Dio.

        I have said who I am and where my profession has been before this damn illness – Private Banking.

        I don’t understand ‘science’ and am feeling rather disillusioned with those who post stuff I don’t understand.

        But thanks for asking. Lovely 🙂

    2. Believe me when I say that these researchers know the association to prostate cancer and ME is not controversial.

      Primers are only one variable in PCR. The fact the Lombardi results were taken from Nested PCR, means that they should have used two sets of primers, but once again they did not. It is therefore totally incorrect for the authors to state that the primers are not one of the reasons for the previous negative study from this group. If they wanted to prove it was not the primers, they should have replicated the original study, and then attempted to alter one variable at a time. Not completely redesign the methodology.

      The studies highlighted as using the same primers, also altered other variables. Again, they all used the VP62 clone. Which would hinder their ability to detect any person who is positive.

      Therefore, no it is not conclusive at all. Lo et al did support the findings of Lombardi et al. They found the same virus.

      This will all be over shortly and we can move on to clinical trials.

  3. I’d say ‘diluted’ and ‘conclusive’ are two different things. As far as I know Mikovits and Lombardi are still doing further research to substantiate their claims and have consistently questioned the methodology of other studies.

    From my point of view I’d wait for the next major press release from them and see how the scientific community receives it. The fact that the initial test launched the scientific community into a frenzy of testing shows you just how seriously they took the initial results.

    So you have to ask yourself why so much interest if it’s such an obvious dead end?

    To be frank, it’s just a conspiracy theory but the question has been raised about whether contaminated vaccines could have carried XMRV into the human blood stream. The oppositional studies to the XMRV link have described the ease of XMRV contamination for instance.

    The scale of public health crisis and the potential for reparations that would be owed could be monumental if XMRV was linked to the actions of national medical institutions.

    That eventuality would compel the medical community to defend itself as much as governments might.

    There were already vested political interests before the issue of culpability is brought into it. (For instance, un-cured disabled people nurse the national unemployment figure, treatment over cure creates industry etc).

    You wonder sometimes if M.E sufferers shouldn’t raise money to buy the services of a PR guru.

  4. This study claims to replicate the methods used by Lombardi et al (the WPI/CC/NCI paper, that kicked off all the “XMRV in CFS” stuff). This is untrue.

    They are saying is that they can’t find any evidence of VP62 with their assays, or any HMLV resembling DG-75. Perhaps they genuinely believe that only VP62 qualifies as XMRV and anything else would not be XMRV at all so there would be no point in looking for it. HIV has 85% variability – 0% variability in a retrovirus is just not credible.

    Lombardi et al set theirs to find VP 35, VP42 and VP62 – in other words a much wider spectrum of the Human Gamma RetroVirus. No study that sets its primers by VP62 alone has found XMRV in ME/CFS.

    “all included patients met the CDC international consensus criteria for CFS [19]. Patients with the Fukuda-specified exclusionary psychiatric disorders, or somatisation disorder (as per DSM-IV), were not included. all included patients met the CDC international consensus criteria for CFS [19]. Patients with the Fukuda-specified exclusionary psychiatric disorders, or somatisation disorder (as per DSM-IV), were not included.”

    They characterized their cohort by Fukuda alone – Lombardi used CCC and Fukuda.

    “Each PCR included six no-template (water) controls and a positive control, plasmid pXMRV, containing the full-length XMRV isolate, vp62, for which we are grateful to R. Silverman.”

    They used water for negative controls again, and calibrated their PCR with the VP62 clone.

    So these two papers on XMRV in CFS involved different diagnostic criteria and different tests to choose the patient cohorts.

    “Considering further the fact that these sera did not behave consistently in both ELISAs, we believe that no specific serological reactions could be detected in the sera of the CFS patients.”

    That says a lot. They both had different results, so there is nothing to see. And if you add a third, fourth, fifth, etc what then? Which assay works and which does not?

    The only thing that can bring us forward is either one of the following:

    1) A negative full-replication study.

    2) Or positive studies, whether they are fully replicating the original one or not (if they are, they will replicate the results (since here we are talking about positive results) if they aren’t, then they are validating the results. In either way, that advances us.

    1. ‘The only thing that can bring us forward is either one of the following:

      1) A negative full-replication study.

      2) Or positive studies, whether they are fully replicating the original one or not (if they are, they will replicate the results (since here we are talking about positive results) if they aren’t, then they are validating the results. In either way, that advances us.’

      I could not agree more Clouty.

      And thanks (again) for posting things I could (almost) get my head around 🙂

      Maybe, finally, perhaps, the things you say will come to pass – in the meantime negative studies dominate.

      Sometimes it is hard to ‘keep the faith’, but then the road was never going to be anything less than rocky was it?

      1. The negative studies do not dominate. They could be seen as being the equivalent of using tests for other viruses to look for MRV’s. Why would anyone assume they would work.

        People don’t realise that the original ME study, Lombardi et al, was backed up by the second study, Lo et al.

        XMRV is a Polytropic/Xenotropic hybrid. It was the same virus.

      2. Do you believe that it is the scoreline that matters? If so, then it’s no wonder that you are struggling to keep up with the science.

        1. Depends from which angle you look at it.

          The negative studies ‘dominate’ the media currently. They also ‘dominate’ in terms of quantity. Quality, accuracy and relevance is another issue.

          The problem here is that in both of your criticisms the context is inferred rather than confirmed.

          1. Don’t worry it will be over soon. Then onto clinical trials. The science is holding up, and the negative studies are only negative. They have produced almost no observations.

  5. I feel wound up when we keep getting all these negative papers from the likes of Wessley & Co., and as they never do a true replication trial it is obvious that they are contriving a realistic-looking work which is nothing of the sort, but is able to take many people in. It strikes me that they are not only unwilling to accept XMRV as associated with ME, but are running scared and are getting more & more desperate in their efforts to decry it.
    My own virology training, 40 years ago, in vet school, was very limited, but I do have over 30 years’ experience in different veterinary fields, including setting up a basic veterinary laboratory in the Himalayan foothills of Nepal, working with Rinderpest in Yemen, and isolating Mycoplasma from goats at the Moredun Institute as part of my M.Sc. I’m still helping my younger son in writing up his college experiments – he’s doing bio-medical science at Brighton.

  6. Desperation from the Government officials. Plenty of spin and light on the facts. Like I have said, it is not long now. It’s going to cause some waves this.

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