Another negative XMRV study – Journal of Emerging Infectious Diseases, February 2011

EID Journal HomeVolume 17, Number 2–February 2011

No Xenotropic Murine Leukemia Virus–related Virus Detected in Fibromyalgia Patients

Joanna Luczkowiak, Olalla Sierra, Jorge Juan González-Martín, Gabriel Herrero-Beaumont, and Rafael Delgado Comments to Author
Author affiliations: Hospital Universitario 12 de Octubre, Madrid, Spain (J. Luczkowiak, O. Sierra, R. Delgado); and IIS-Fundación Jiménez Díaz, Madrid (J.J. González-Martín, G. Herrero-Beaumont)

Suggested citation for this article

To the Editor: Xenotropic murine leukemia virus–related virus (XMRV) is a recently described human retrovirus that has been associated with prostate cancer and chronic fatigue syndrome (CFS) (1,2). XMRV is similar to a classic murine endogenous leukemia retrovirus, murine leukemia virus (MLV), which infects strains of mice that do not express the specific viral receptor. XMRV is genetically close to, although differentiable from, MLV. The first evidence of its presence in humans was obtained by Urisman et al. in prostate cancer tissue (1). In 2009, Lombardi et al. (2) found XMRV sequences and specific antibody responses in 67% of a large group of patients with CFS in North America. This association was notable because XMRV sequences were found in only 4% of healthy controls. These results have generated controversy because several independent studies, mainly in Europe (3–5) but also in North America (6), have been unable to detect XMRV sequences in patients with CFS. Furthermore, a recent report from North America (7) appears to confirm the initial results by Lombardi et al. (2) in patients with CFS and expands the viral association to a wider variety of XMLV-related viruses that seem closer to polytropic mouse endogenous retroviruses.

Fibromyalgia is a multifactor condition characterized by widespread pain and diffuse tenderness. Although trauma and stress can worsen or even precipitate development of the syndrome, infections with certain viruses, including hepatitis C virus and HIV, have been associated with development of fibromyalgia (8). Nevertheless, fibromyalgia remains a disease of unknown etiology. Although CFS is a distinct entity, features shared by both diseases suggest that CFS and fibromyalgia represent the same underlying condition (9). Additionally, because they are often accompanied by a noticeable mental health effect (9), the presence of a potential neurotropic retroviral agent in both diseases could explain these similarities. Therefore, we studied the presence of XMRV and polytropic MLV–related retroviruses in a group of patients with fibromyalgia.

During January 2010, blood samples were collected from 15 patients in whom fibromyalgia had been previously diagnosed according to American College of Rheumatology criteria (www.rheumatology.org/practice/clinical/classification/fibromyalgia/1990_Criteria_for_Classification_Fibro.pdf). Ten healthy blood donors served as controls. For XMRV screening, we used DNA extracted from 400 μL of whole blood collected in EDTA tubes by the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). Nested PCR was done by using 5 sets of primers corresponding to the gag (3) and env (2) regions of XMRV as described (2,3,7). The first round of PCR was conducted by using 500 ng of genomic DNA, equivalent to 7.5 × 104nucleated blood cells, in a final volume of 50 μL, by using the Expand High Fidelity PCR System (Roche Applied Science, Basel, Switzerland). A second round of PCR was conducted under the same conditions by using 5 μL of the first reaction product. Details of the nested-PCR strategy were as follows: gag region was amplified by outer primers 419F and 1154R (2) and 3 sets of inner primers: XMRV-FI-441/XMRV-RI-566 (3), MLV-GAG-I-F/MLV-GAG-I-R, and MLV-NP116/MLV-NP117 (7). Nested PCR for env was performed by using outer primers 5922F and 6273R (2) and 2 sets of inner primers: 5922F/6173R and 5942F/6159R (7). Primers for human β-globin were used as positive controls of human DNA amplification (3). The full-length molecular viral clone VP62 (obtained through the National Institutes of Health AIDS Research and Reference Reagent Program [Rockville, MD, USA] from R.H. Silverman and B. Dong) (10) was used as a positive XMRV control. All samples were examined on a 2% agarose gel stained with ethidium bromide (Figure). The overall sensitivity of the nested PCR procedure, estimated by spiking VP62 into negative samples, was 1–10 copies per sample.

Using highly sensitive PCR tools and a multiple set of primers to detect xenotropic and polytropic MLV–related sequences, we found no evidence of MLV-related sequences in blood cells from fibromyalgia patients or controls. Our results agree with those from studies of CFS cohorts in Europe and North America that also failed to confirm XMRV in blood samples (3–6). Technical issues or geographic specificities probably could not account for such a difference; therefore, these negative results raise concerns about the role of XMRV in these syndromes. Nevertheless, with this relatively small population we cannot absolutely exclude an association of XMRV or polytropic MLV–related viruses with fibromyalgia. However, a proportion of fibromyalgia cases with XMRV >22% would be unlikely (3/15 cases, 95% confidence interval 0–3), which is clearly insufficient to support a significant association between XMRV and fibromyalgia.

Fibromyalgia does not appear to be associated with XMRV or polytropic MLV–related viruses. The role of these new agents in human disease, and specifically in CFS, remains to be clearly confirmed in multicenter and standardized studies.

Acknowledgment

This study was supported by grants Fondo de Investigación (FIS)-PS09/01625 to G.H.-B. and Fundación para la Investigación Prevención del SIDA en España 36749, FIS-PI080806, and European Union Seventh Framework Programme CARMUSYS PITN-GA-2008213592 to R.D.

References

  1. Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, et al. Identification of a novel gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog. 2006;2:e25. PubMed DOI
  2. Lombardi VC, Ruscetti FW, Das GJ, Pfost MA, Hagen KS, Peterson DL, et al. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science. 2009;326:585–9. Epub 2009 Oct 8. PubMed DOI
  3. Erlwein O, Kaye S, McClure MO, Weber J, Wills G, Collier D, et al. Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome. PLoS ONE. 2010;5:e8519. PubMed DOI
  4. Groom HC, Boucherit VC, Makinson K, Randal E, Baptista S, Hagan S, et al. Absence of xenotropic murine leukaemia virus–related virus in UK patients with chronic fatigue syndrome.Retrovirology. 2010;7:10. PubMed DOI
  5. van Kuppeveld FJ, de Jong AS, Lanke KH, Verhaegh GW, Melchers WJ, Swanink CM, et al. Prevalence of xenotropic murine leukaemia virus–related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort.BMJ. 2010;340:c1018. PubMed DOI
  6. Switzer WM, Jia H, Hohn O, Zheng H, Tang S, Shankar A, et al. Absence of evidence of xenotropic murine leukemia virus–related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States.Retrovirology. 2010;7:57. PubMed DOI
  7. Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, Wang R, et al. Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors. Proc Natl Acad Sci U S A. 2010;107:15874–9. PubMed DOI
  8. Buskila D, Atzeni F, Sarzi-Puttini P. Etiology of fibromyalgia: the possible role of infection and vaccination. Autoimmun Rev. 2008;8:41–3. PubMed DOI
  9. McKay PG, Duffy T, Martin CR. Are chronic fatigue syndrome and fibromyalgia the same? Implications for the provision of appropriate mental health intervention. J Psychiatr Ment Health Nurs. 2009;16:884–94. PubMed DOI
  10. Dong B, Kim S, Hong S, Das GJ, Malathi K, Klein EA, et al. An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors. Proc Natl Acad Sci U S A. 2007;104:1655–60.PubMed DOI

12 thoughts on “Another negative XMRV study – Journal of Emerging Infectious Diseases, February 2011”

  1. Too small a study to be significant (15 patients), and not on ME.

    It didn’t even have a normal publication process, as it is a letter to the editor. They used whole blood which Lombardi et al. did not. Used Nested PCR, which Lombardi et al. did not. They used RT-Nested PCR. They used VP62 to calibrate their assay, which Lombardi et al. did not use, and not a positive clinical sample.

  2. O cool another negative.

    No doubt any comments I make will be torn asunder by those claiming a better understanding of the ‘science’.

    How many negatives will it take I wonder?

    1. I don’t understand the science either, but logic would dictate that the accuracy and method of the test are of equal or greater importance than the quantity of tests.

      I find if I don’t know anything about the technical side of a topic I can at least reference what is said next to the different types of logical fallacy.

      Using quantity to affirm a conclusion is a form of:- http://en.wikipedia.org/wiki/Argumentum_ad_populum

      There does seem to have been some questions raised towards the methodology that I have yet to see a response to. So while I harbour no blind optimism of a correlative link between M.E and XMRV, I have to remain open minded for the time being.

  3. This is the second Spanish study. The first was a small one out of Can Ruti Hospital, Barcelona, with around 15 patients. It was funded with help from patients, and Mikovits showed them the techniques necessary. It was positive, they found XMRV in similar percentages to the other positive studies, but the study group are not able to access further funds to continue their work. This study has failed to find a publisher so far, as has the WPI UK Study.

    This second one was publicly funded. Like all the other negative studies we have seen, finding publication presents no problems.
    One must ask why it is that so many positive studies have difficulty finding , publication, while negative studies get straight into print, sometimes as little as a few days after submission. Don’t say it is down to the quality of the studies involved, because we have seen how very poor the negative studies have been. Unless poor study design is a prerequisite for publication :p

    Remember the CDC and the Lo/Alter studies, last summer?

    Back to this latest Spanish study. The thermodynamic task of locating a viral sequence integrated in DNA and merely mixed with DNA is very different and the parameters involved in the chemical reaction have to be changed accordingly. This needs DNA from a known clinically positive sample. As we know, the WPI offer to supply a known positive sample.

    The technique of calibrating PCR conditions to locate a synthetic clone simply does not work and would defy the known laws of thermodynamics.

    Continuing to use a technique with a 100% failure rate is beyond incompetent. Good scientists copy successful techniques, not unsuccessful ones. The people who failed to find the virus all used variations of the same technique which has always failed. Why test for XMRV using techniques which are now known not to work?

    Again, they calibrate with an artificial clone as have all the other negative studies.

    Danielson, 2010 and Klimas 2010 both have laid out clearly the problems of finding XMRV and it’s family of HGRV’s. Yet so many study designs fail to take notice.

    1. I do have a tendency, as you know Jace, to repy to posts that address my concerns, so I just wanted to say, thanks.

      If what you have said is in anyway wrong, I know that someone will come along and further enlighten me.

      Now that is what I like about places like this.

  4. I wouldn’t be concerned by this. It is a letter to the editor, only looked at 15 people with Fibro, not ME, and used a different methodology to Lombardi et al. Primers are not the only thing that can be changed when using PCR.

  5. Hi Currer,

    I don’t. I think it ‘fits’ quite nicely actually. I just don’t profess to have the knowledge that others’ do about the science, and abhor references to conspiracy theories.

    Fundamentally though, if XMRV was the big nasty it is sometimes rumoured to be, then I honestly think we would know by now – and we don’t.

    XMRV will be and should be the focus and if any ‘link’ is subsequently proven, then so be it. I shall deal with that when I have to – it isn’t denial on my part, I just remain more open-minded than some whose dictat dominates some forums.

    Thank you for asking.

  6. I too think it fits nicely, but I think you underestimate the length of time a genuinely new discovery takes to be accepted and the acrimony of the debate in the scientific community while this happens. We would not necessarily know by now.

    Your posts have elicited some very informative responses from those who have either the background to understand the details of this science or who have taught themselves to understand it so that they can evaluate the papers. This has made for some very interesting and informative reading on this site and I have benefited from following this debate.

    For me the most revealing part of the WPI’s work has been the data on the transmission of this virus in families. This fits my family and makes sense of a lot of ill-health that could not be seen as a coherent whole before. Even if some details of the science are incorrrect I feel sure the WPI are on to something.

    I have watched how this illness has spread down the generations during the time I worked for one of the local ME Groups.
    It became increasingly common in children at puberty wheras in my generation it affected the 20-40 age group. Even my GP has noticed this.

    Yet the DOH have never commissioned an epidemiological survey on ME. We have no figures on how many it affects in this country, how it spreads, whether its pattern of spread is changing over time. We do not know what happens to those who have been ill for many years – do they develop other diseases? And this despite repeated calls over twenty years from patient organisations and their MPs for just these surveys.

    You would think they would be worried. I wish I could be as certain as you on the conspiracy theory.

  7. It’s not a conspiracy exactly, just a case of people protecting their turf and self interest combined with laziness, lack of imagination, and the unwillingness on the part of major players to accept any egg on their faces.

    If only independent research was funded, including at the WPI, we could move forward more rapidly, but when people who hold the purse strings are heavily invested in the NICE guidelines view of Myalgic Encephalomyelitis, the lack of funding holds back progress.

    Not a conspiracy exactly, more selfish self interest and lack of imagination.

    1. Howdy Jace,

      I was under the impression that the WPI had raised in excess of US$6 million from private donations.

      Is that not sufficient to better substantiate their fundamental claims?

      I still maintain that those whose commercial interests have benefitted from HIV and AIDS would be all over XMRV like – well – a rash.

      They most probably are… Drugs are certainly already developed or in development specifically to target XMRV.

      What exactly XMRV does in animals and then humans, i.e. investigations into causation, would surely have begun as soon as the retrovirus was discovered. Potentially this is much bigger than merely CFS/ME and our suffering.

      That is one of the reasons why I remain on the fence – despite the pain that causes – over any research purporting a link. Particularly, as there is nothing to explain causation in sufferers like myself.

      It just doesn’t ‘add-up’ for me – yet…

      And all this focus on the WPI because the link seems to ‘fit’ takes focus and money away from other projects and research, which in the absence of causation, is, in my view, more vital.

      Just an opinion. Everyone has their own and they are of course entitled.

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