XMRV Controversy Heats Up — Two Important New Studies

As expected, the XMRV virus research has stimulated interest in a host of studies to see if the initial study results can be replicated and to discover treatments for XMRV. Since our last newsletter gave updates on XMRV testing and research, two interesting new studies have been reported.

The first study suggests that of the 10 most common antivirals used against retroviruses, the only one that showed any effectiveness against XMRV (in test tubes) was AZT. This will give researchers an important starting point in exploring treatment for XMRV. I would NOT recommend this treatment yet for CFS though, as it can be fairly toxic and we don’t know if it will have any benefit.

The second study was done in the UK, and screened 186 CFS patients for XMRV using PCR testing (looking for genetic material). None of these patients tested positive for XMRV. This finding should be tempered by the same issue being found for prostate cancer and XMRV. Where the American studies found XMRV to be more common in prostate cancer, this association was not found in other European studies. This suggests that either XMRV infection is more of an issue in the US or that the testing methodology used in the European study was inadequate (which is what the WPI is claiming).

As a medical reporter who has been closely involved in the CFS & fibromyalgia field as a patient, physician and researcher for over 30 years, I find both the science and politics to be fascinating. You will hear some hard hitting attacks (sometimes done subtly, sometimes not so subtly) coming from both sides. And I suspect the melee has just begun.

In the following we will present the two above studies in more depth. In addition, we’ll look at initial reader comments on treatment from our community bulletin board, and look at the rebuttal of the UK findings by the WPI (which did the initial study) and some background on a few of the new study authors — significant, as one of them seems determined to have CFS be treated as a psychological problem!
What Do the Two New Studies Show?

The first study used test tube experiments to see which antivirals might be effective against XMRV. The researchers took the 10 most common AIDS drugs (AIDS and the XMRV virus are both retroviruses) and tested their effectiveness against XMRV. I found that I was holding my breath while waiting to get the answer to the question of what might work against XMRV. The results?

Of the 10 antivirals, the only one that showed any effectiveness against XMRV was an old antiviral called AZT ("Retrovir" being the brand name and the generic name being "zidovudine"). This medication has been around long enough that it is no longer under patent protection. This has the benefit of making the medication less expensive but also has the downside of making it less attractive for drug companies to spend research dollars on.

I would not recommend trying this medication at this time. Although it may become worth using if shown to be effective in chronic fatigue syndrome, without evidence of its effectiveness for this disease the medication is too toxic. To give you an idea of the concerns, here's the FDA-required black box warning in the Physician's Desk Reference under AZT (Retrovir/zidovudine):

WARNING

Retrovir (zidovudine) has been associated with hematologic toxicity including neutropenia and severe anemia particularly in patients with advanced human immunodeficiency virus (HIV) disease.

Prolonged use of Retrovir has been associated with symptomatic myopathy.

Lactic acidosis and severe hepatomegaly with steatosis, including fatal cases, have been reported with the use of nucleoside analogues alone or in combination, including Retrovir and other antiretrovirals.

To put it in perspective though, AZT has been used safely in an enormous number of AIDS patients.

The bottom line? Although it may be helpful, and I consider this research study to be very important as a first step, I would not recommend being the first CFS patient on your block to try it. I would wait and let other people volunteer to be the guinea pigs. If we find that many people report it to be helpful, we will move this discussion to the next level.
What Does the Second Study Show?

Basically, as noted above, it performed PCR testing on 186 people in England suffering with CFS and found that none of them was positive for the XMRV virus. So how does one make sense of this conflicting information?

There are several probable answers to what is occurring:
1.


It could simply be that the testing methods used in the second study were not reliable. This is the impression of the folks at the WPI, where the initial study was done, and in a press release the WPI comments on the new study.

This is not an unlikely scenario, as testing done at the WPI was much more complex and I suspect more likely to be accurate.
2.
XMRV virus may simply be more common in humans in the United States and rare in Europe. This was supported by the four studies done on XMRV and prostate cancer.
3.
Questions have been raised about the patients used in both studies.

One of the main researchers in the England study is Dr. Simon Wessely, a nice and very bright fellow who I suspect is so blinded by his determination to have CFS viewed as a psychological disease requiring only counseling that I find most of his research to have a decidedly unhealthy bias. I think if he applied his same bias to patients dying of lung cancer, he would also see them as being “not really sick” (i.e., he would see them as crazy). On the other hand, I have enjoyed the work of Anthony Cleare on HPA axis/adrenal issues in CFS over the years. I find it interesting that they have teamed up on a number of studies recently — including this one.

The source of the WPI study patients, based on the paper, is also not clear to me. My main concern is that they not come largely from a single epidemic of CFS, such as the Tahoe group, as this could largely skew the results. Again, we look forward to seeing the results of testing of patients from around the country (and indeed around the world) to see what percentage are positive.

For those setting up studies looking for XMRV in CFS patients, the above study suggests a simple way to answer these questions if they arise again. As a medical reviewer, it would be very helpful if the studies sent all or even 10-20% of their blood samples to the WPI for testing, while also doing the testing in their own labs. Half of the samples sent to the WPI should be from healthy patients, and the samples should be blinded (i.e., not labeled as to whether they are coming from CFS or healthy control patients). If the samples sent to the WPI clearly again come up positive for CFS and negative for healthy patients, as in the earlier study, it would immediately answer the question of whether there is a methodology problem and it would quickly answer many of our questions. So in real life, there is really a very simple approach that could answer these questions.

In the mean time, I suspect the debate will continue to heat up. Stay tuned!

I agree with Dr. T's logic and data. Several ppl were upset by Vancerd's recovery on the ProHealth forum...like a disbelief or envy. Vancerd's root cause was gallstones in the liver and is still feeling great and sleeping well after several months now.

Another great study would be to "treat" the XMRV + CFS patients and verify a percentage of complete recovery. It is known that Hyperbaric Oxygen treatment (HBOT) kills off every known infection above 2.6 Atmospheres of pressure. The treatment is very expensive BUT might be the best way to study XMRV as "the root cause". (1)

Still another root cause might be toxic metals. Lead is an antagonist of calcium and can disrupt bone marrow white blood cells. The hypothalamus has an affinity for mercury. (2)

(1) Hyperbaric Oxygen Therapy, Neubauer MD, Walker
The Oxygen Revolution by Dr. Paul Harch
www.hbot4u.com

(2) No More Amalgams, Andrew Cutler PhD (professor of pharmcokinetics)

VESTED INTERESTS OF SIMON WESSELY

One of the co-authors of this study, Simon Wessely, has a vested interest in the outcome of such virus studies. Simon has staked his entire academic career on the idea that neurological diseases like chronic fatigue syndrome are caused by purely psychological factors.

Therefore Simon Wessely would be the last person you would want in a study like this: if he finds the virus in CFS patients, he shoots himself in the foot, and his career may never recover. So I wonder just how hard he looked for this XMRV virus.

The odd thing is, the authors declared in this study that they have no competing interests: not true! It is very much in the interest of Simon Wessely to not find this XMRV virus.

Disturbing negative results from the latest replication study.

Second XMRV Negative Study …
Still In Search of a Proper and Robust Replication Study

Suzanne D. Vernon, PhD
Scientific Director
The CFIDS Association of America



A second study looking for XMRV in CFS patients has come up empty-handed. On February 15, 2010, authors from the United Kingdom (UK) published a paper in the open access journal Retrovirology titled, “Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome.” These investigators used quantitative and sensitive methods to look for XMRV in blood samples collected from CFS patients, healthy blood donors and clinic patients with other disorders. They also looked for antibodies that could block XMRV from infecting cells in the liquid portion of the blood (serum and plasma) from CFS and controls.

Before getting to the details of the paper, let’s look at the group that reported these results. The lead investigator, Dr. Harriett CT Groom, and three other authors are from the department of virology at Medical Research Council National Institute for Medical Research, the UK’s equivalent to the United States’ National Institutes of Health (NIH). One of these investigators,Dr. Jonathan Stoye, was the co-author of the editorial in Science that accompanied the report from Lombardi, et al, that first made the association between XMRV and CFS. Dr. Stoye is a world renowned retrovirologist. Four authors are from the CFS group at St. George’s University of London, including Dr. Jonathan Kerr, best known for his research describing the genomic and infectious aspects of CFS. Dr. Kerr is also a co-investigator on the Whittemore Peterson Institute’s grant funded by the NIH. Two authors, including Dr. John Gow, are from Caledonia University in Glasgow Scotland. Dr. Gow has a long publication record in CFS research. Two other authors come from Barts and the London National Health Service Trust and University College in London. Each of their contributions to this study are described at the end of the paper.

Blood Primer:

Red blood cells (erythrocytes): Contain hemoglobin and help distribute oxygen to cells.

White blood cells (leukocytes): Perform functions of the immune system.

Platelets (thrombocytes): Perform clotting functions and prevent bacteria from entering cells.

Plasma: Represents about 55% of the blood fluid and is comprised of water, platelets and blood cells. If you extract clotting proteins from plasma, this is called serum.

There are many different methods used for collecting, storing and processing blood samples. Components of the blood may be tested for different things, and tests of different blood components may yield different results when tested for the same thing. All of these factors must be accounted for in standardizing blood tests over time by the same laboratory and across different laboratory settings. In research, it is common practice to use samples collected and stored over time; however, variances in protocols for collection, storage, shipping and processing must be considered in data analysis and conclusions.

This study included three different cohorts:

1. St. George’s University of London (SGUL): This cohort was comprised of 142 adult CFS patients and 157 healthy blood donors from the CFS Group in the Division of Cellular & Molecular Medicine. These subjects were 18 to 65 years of age. Blood samples containing both cell and liquid fractions were collected between 1.5 and 4 years after a CFS diagnosis.
2. Barts and the London National Health Service Trust (BLT): This cohort comprised 226 serum samples taken in 2008 and 2009. Fifty-seven were obtained from patients in the prenatal clinic; 58 were taken from patients with blood disorders; 55 came from liver patients and 56 were from the kidney clinic. These samples were all used as control samples.
3. Glasgow Caledonian University (GC): This cohort comprised 28 CFS patients (20 sera and 8 plasma samples) and 12 controls (8 sera and 4 plasma samples). CFS patients were ages 28 - 79 and samples were collected between 1995 and 2003.

The authors report that all CFS patients met 1994 (Fukuda) CFS case definition criteria, but they make no other statements about the characteristics of those patients in terms of disease severity of other clinical markers. The patient cohort being comprised or patients from different centers is not unique to this paper; samples from the CFS patients in the Science paper were gathered from several regional physicians’ practices, according to information on the Whittemore Peterson Institute’s website.

The investigators led by Dr. Groom reported several different experiments in the Retrovirology paper. They used polymerase chain reaction (PCR) methods identical to those reported by Lombardi et al, in the Science paper, yet they were unable to detect XMRV in the samples from the SGUL samples. They then developed a different, but very sensitive, quantitative PCR assay that could detect as few as 16 copies of XMRV DNA. Still there was no evidence of XMRV nucleic acids in the SGUL samples. They used an appropriate technique called “spiking” to show that nothing was present in the samples that would prevent XMRV from being amplified.



Neutralizing antibodies prevent viruses from entering a cell, thereby “neutralizing” the infection. Groom et al, looked for evidence of neutralizing antibodies in the sera or plasma from all patients and controls. They found 26 (4.6%) of the 565 serum samples could neutralize XMRV, preventing it from entering the cell. However, only one of the 26 was a serum sample obtained from a CFS patient. Most of these were also able to neutralize response to other similar viruses, indicating significant cross-reactivity in serological responses.

The Retrovirology study found evidence of XMRV by detecting specific antibodies in samples from study subjects, demonstrating that XMRV has infected people. But like the other study from the U.K. reported in PLoS ONE, this study did not detect XMRV at a higher rate in people with CFS. Why? The PCR technique used in the paper was identical to the Science paper and other methods they used could be considered better and more sensitive. The antibodies used by the two labs were different, though. These investigators did not test quite as many healthy control blood samples (157 in the SGUL cohort) as did the authors of the Science paper (218 healthy controls), which found XMRV in 3.7% of healthy controls. (The source of healthy control samples was not described in the Science paper.) Perhaps more important is that this Retrovirology paper tested for XMRV in healthy blood donors; blood donors are screened for many things and are likely to be a much healthier group than controls obtained from the general population. The inclusion of the BLT comparison group of individuals with other conditions was new for this study.

Why wasn’t XMRV DNA found in the CFS samples? One difference between the CFS patients selected for the SGUL cohorts and those tested in the Science paper might be severity and duration of illness. The SGUL CFS cohort blood samples were taken relatively early in course of CFS (1-4 years). The CFS patients in the Science paper were severely ill in addition to having immune dysfunction, although specific characteristics have not been released. It could simply be that the CFS patients in the SGUL cohorts were not comparable (e.g., as sick as long) to those studied in the Science paper. However, Dr. Kerr’s reputation and experience in CFS/ME research, along with his current collaborations with the Whittemore Peterson Institute, suggest that he would have taken care to use similar selection criteria in his design of the study. (It is noted in the acknowledgements that “JK” – Jonathan Kerr – was one of the three authors who conceived of and designed the study.)



But if XMRV is able to infect people – as shown in this paper, in the Science paper and in the prostate cancer papers – shouldn’t experts be able to detect the virus or find antibodies to the virus? Not necessarily. Viruses need to survive. The best way for them to do this is to adapt to the host so that they can persist. Staying at very low levels is one way to evade detection by the immune system in order to survive. Even though Dr. Groom and colleagues developed a sensitive and quantitative PCR test, XMRV may be able to hide out with fewer than 16 copies (the limit of this paper’s sensitivity), or may not be present in the blood cells of shorter-duration CFS patients. Another trick viruses use is to sabotage the immune response. In an elegant study published in the Proceedings of the National Academy of Sciences in February 2010, French investigators showed that retroviruses, including XMRV, contain an immunosuppressive domain (ISD) in envelope protein. The envelope is the outside portion of the virus that is the immune response is supposed to recognize. This ISD allows the virus to infect the cell but somehow – and this mechanism is not understood – prevents the body from mounting an immune response and developing neutralizing antibodies to the virus. This newly discovered viral “trick” could explain why so few serum samples had neutralizing antibodies to XMRV in the experiments described in the Retrovirology study. The high rates of XMRV antibodies described in the Science paper might be explained by serological cross-reactivity. There are known cases where the immune system comes into contact with a complex agent like a virus, it stimulates multiple reactions to parts of the virus and other molecules.

So what is it going to take to get a proper and robust replication study done? Intense attention to detail. Because patient selection has been a potential confounding factor in the two negative studies, the scientific community must understand the clinical characteristics of the CFS patients who were XMRV positive in the Science paper. CFS is a chronic, heterogeneous illness with many possible subtypes, each made even more complex by common co-morbidities such as irritable bowel syndrome, fibromyalgia and depression. The lack of universal subtypes or staging criteria for severity and duration of illness makes comparisons challenging. Many CFS patients undergo a variety of treatments in order to get relief from this debilitating disease. Treatments, especially those that directly act on the immune system, may also affect the life cycle of XMRV and detection of the virus.

Standardization of testing methods must also progress. As we reported in January, the U.S. Department of Health and Human Services Blood XMRV Scientific Research Working Group is developing analytical panels that will allow multiple laboratories to standardize methods to optimize sensitive detection of XMRV proviral DNA and viral RNA. Once methods are standardized, these same laboratories plan to test coded panels of blood samples obtained primarily from healthy blood donors and from CFS patients who have been reported to be positive for XMRV.

Until methods are standardized and the scientific community is provided information about the specific characteristics of the CFS subjects (and controls) who tested positive in the Science paper, be prepared to read more negative studies. Hopefully the Science investigators will make this information available before interest in XMRV being associated with CFS fades and becomes yet another foiled attempt at solving CFS. Achieving scientific consensus on the role of XMRV in CFS certainly warrants more research and greater collaboration, as do so many other important discoveries being made.

Citations:

Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome. Groom HCT, Boucherit VC, Makinson K, Randal E, Baptista S, Hagan S, Gow JW, Mattes FM, Breuer J, Kerr JR, Stoye JP, Bishop KN. Retrovirology: 15 February 2010. 10.1186/1742-4690-7-10

Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Lombardi VC, Ruscetti FW, Gupta JD, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. Science 8 October 2009. 1179052.

Supporting online material for Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Lombardi VC, Ruscetti FW, Gupta JD, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. Science 8 October 2009.

Erlwein O, Kaye S, McClure MO, Weber J, Willis G, Collier D, Wessley S, Cleare A. (2010) Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome. PLoS ONE 5(1):e8519. doi:10.1371/journal.pone.0008519

A new virus for old diseases? Coffin JM and Stoye JP. Science 8 October 8 2009.

Retroviral infection in vivo requires an immune escape virulence factor encrypted in the envelope protein of oncoretroviruses. Schlecht-Louf G, Renard M, Mangeney M, Letzelter C, Richaud A, Ducos B, Bouallaga I, Heidmann T. Proc Natl Acad Sci U S A. 2010 Feb 8.



Suzanne D. Vernon, PhD Scientific Director

Suzanne D. Vernon, PhD, earned her doctorate in virology at the University of Wisconsin at Madison and worked in public health research on infectious diseases at the U.S. Centers for Disease Control and Prevention for 17 years before joining the CFIDS Association of America’s staff as scientific director in 2007. She has more than 70 peer-reviewed scientific publications on topics including human immunodeficiency virus, human papillomavirus, cervical cancer and chronic fatigue syndrome. Dr. Vernon has initiated and participated in numerous international and multidisciplinary research collaborations and she now leads the CFIDS Association’s research program. The CFIDS Association of America is the nation’s largest philanthropic supporters of CFS research.

Dr. Vernon analyzes the third study that failed to link XMRV to CFS.


Playing A Weak Hand Well

Suzanne D. Vernon, PhD
Scientific Director
The CFIDS Association of America



XMRV was not detected in a third follow-up study from a well-characterized cohort of CFS subjects. In a paper published on February 25, 2010 in the British Medical Journal (BMJ) titled, “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,” Frank van Kuppeveld and an expert team of microbiologists and clinical investigators failed to find evidence of XMRV. This team used a sensitive polymerase chain reaction (PCR) test that could detect as few as 10 copies of XMRV in 100,000 peripheral blood mononuclear cells (PBMCs) to test PBMCs from 32 CFS patients and 43 controls.

The CFS cases were enrolled from 1991 to 1992, satisfied Oxford Criteria (published in 1991), were severely ill with debilitating fatigue of at least one year, and reported duration of symptoms ranging from 2 to 45 years. The controls were neighbors selected by the CFS subjects, the same sex as and within two years of age of the CFS subject. This study used a well-characterized cohort, well-processed and preserved blood samples and a sensitive technique. Its weakness is the small sample size. Use of the less-restrictive Oxford criteria may be objectionable by current standards, but samples were collected before publication of either the Fukuda research criteria (1994) or the Canadian clinical definition (2003). So far all the XMRV studies have used banked CFS samples.



With the third negative study published in just 51 days, it is now harder to explain the negative results based on CFS patient characteristics and methods alone. The implication is that XMRV is likely to explain a subset of CFS rather than all cases defined as CFS (using any of the seven existing definitions). The PLoS ONE paper by Erlwein, et al, the Retrovirology paper by Groom, et al, and now the van Kuppeveld, et al, paper in BMJ all studied well-characterized patient cohorts that met accepted and widely used CFS case definition criteria. Importantly, many – if not most – of the CFS patients selected for these XMRV studies have been the subjects of other CFS studies by experienced investigators. While the CFS subjects from these three studies may be different from the CFS subjects in the Science paper by Lombardi, et al, there certainly must have been some overlap between the cohorts chosen. Put another way, it is unlikely that case definition criteria alone accounts for the discrepant results ranging now from three negative studies to one study that found 67 percent of the CFS patients and 4 percent of healthy controls to be positive for XMRV.



The authors of the Dutch study go into a fair amount of detail to describe why they think their methods do not account for differences in the study’s outcomes compared to the original XMRV study. Here is a brief, yet still pretty technical summary of their methods: they performed a real-time PCR test with the same primers as the original study. Positive and negative controls were included in each of the runs and they used a 22Rv1 XMRV-positive prostate cancer cell line to ensure that they could detect positives. They used a similar amount of nucleic acid as the original study to hunt for XMRV. The samples from all the CFS patients and controls tested negative for two different XMRV genes encoding integrase and gag proteins.

History has taught us the absolute importance of impeccable study design in looking for infectious agents as a possible cause of CFS. Back in 1991, Elaine DeFreitas, PhD, and colleagues published in the prestigious Proceedings of the National Academy of Sciences that they had detected retroviral sequences related to human T-lymphotropic virus type II (HTLV-II) in two-thirds of 31 CFS patients, compared with zero positive findings in 20 controls. This report was not confirmed by other investigators who found the same rate of these HTLV-II-like sequences in controls and CFS cases and the search for viral markers chilled for several years thereafter.

The study by Lombardi et al, published in Science showed that XMRV could be detected in samples from the WPI repository. Unlike the negative XMRV studies where results and characteristics of these CFS cohorts have been the subject of numerous earlier publications, little is known about the patient samples stored in this repository. The Science paper’s supplement refers to a variety of abnormalities without defining how they were measured or how uniformly they show up among the patients whose samples are stored there. The supplement makes reference to DeFreitas’s PNAS publication. Does this mean that WPI tested the same samples as DeFreitas, et al, or that they were obtained from the same cluster outbreaks mentioned in her paper? We do not know. We also know nothing about the controls reported in the Science paper from the published literature. Because the study design and the methods provided in the Science paper and supplemental material are lacking in detail, it makes it challenging for any investigator, no matter how expert, to replicate this study. It’s not plausible to expect that researchers will piece together information about case selection or laboratory methods posted only to websites or delivered in presentations given to lay audiences to design the methods for serious replication or validation studies.

Since reliable, consistent information about the Science cohort has not been forthcoming, I have carefully analyzed data provided in the Science paper, its supplement and public presentations by two of the authors. The WPI investigators conducted a number of assays to detect XMRV DNA, protein, infectious virus and antibodies against XMRV. I used the patient ID numbers provided in the paper to track results. Of the 101 CFS subjects reported in the paper, results for the various assays are shown for only 32 CFS subjects. Of the 32 CFS subjects whose results for any of the tests are displayed, 12 CFS subjects were positive for XMRV on more than one assay. The other 20 CFS subjects were documented as positive by just one testing method. Using information from a public presentation at the federal CFS Advisory Committee, five of the 12 CFS subjects (WPI1169, 1118, 1150, 1199 and 1125) included in the Science paper were also reported to have cancer – either lymphoma, mantle cell lymphoma or myelodysplasia. This once again raises questions about the lack of detailed clinical characteristics of the CFS subjects included in the Science paper, and the differing public reports about where the samples originated.

XMRV is a newly described virus that infects humans. There may be virus variability making it difficult to detect, and it may be more easily found in organ tissues rather than blood. There are going to be numerous technical, biologic and epidemiologic challenges associated with linking XMRV to CFS and other diseases including prostate cancer. Whether XMRV is in any way associated with CFS will be the subject of further investigation. But these investigations must be designed appropriately and impeccably. Investigators from several U.S. institutions reported outcomes from recent XMRV studies at the 17th Conference on Retroviruses and Opportunistic Infections (CROI 2010), including infecting and establishing chronic XMRV infection in rhesus monkeys. Interestingly, XMRV was localized to the reproductive and lymphoid organs in these animals.

Time and precious resources are being consumed by studies in which the results are controversial ones. The CFIDS Association of America is working diligently to foster the type of well-designed studies of CFS and XMRV that will provide definitive grounds for moving forward on this hypothesis so that history does not repeat itself. True to our mission statement, we will continue to lead, support and conduct research with integrity, innovation and purpose in order to make CFS widely understood, diagnosable, curable and preventable.

Citations:
van Kuppeveld FJ, de Jong AS, Lanke KH, Verhaegh GW, Melchers WJG, Swanink CMA, Bleijenberg G, Netea MG, Galama JMD, van der Meer JWM. 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. British Medical Journal 2010 (Published 25 February 2010)

Groom HC, Boucherit VC, Makinson K, Randal E, Baptista S, Hagan S, Gow JW, Mattes FM, Breuer J, Kerr JR, Stoye JP, Bishop KN. Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome. Retrovirology. 2010 Feb 15;7(1):10.

Erlwein O, Kaye S, McClure MO, Weber J, Wills G, Collier D, Wessely S, Cleare A. Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome. PLoS One. 2010 Jan 6;5(1)

Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science. 2009 Oct 23;326(5952):585-9.

Lombardi VC, Ruscetti FW, Gupta JD, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. Supporting online material for Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 8 October 2009.

DeFreitas E, Hilliard B, Cheney PR, Bell DS, Kiggundu E, Sankey D, Wroblewska Z, Palladino M, Woodward JP, Koprowski H. Retroviral sequences related to human T-lymphotropic virus type II in patients with chronic fatigue immune dysfunction syndrome. Proceedings of the National Academy of Sciences USA. 1991 Apr 1;88(7):2922-6.

Dr. Daniel Peterson, reporting at the DHHS CFS Advisory Committee meeting on Oct. 29, 2009. http://www.hhs.gov/advcomcfs/meetings/p ... v_cfs.html, accessed Feb. 26, 2010.



Suzanne D. Vernon, PhD Scientific Director



Suzanne D. Vernon, PhD, earned her doctorate in virology at the University of Wisconsin at Madison and worked in public health research on infectious diseases at the U.S. Centers for Disease Control and Prevention for 17 years before joining the CFIDS Association of America’s staff as scientific director in 2007. She has more than 70 peer-reviewed scientific publications on topics including human immunodeficiency virus, human papillomavirus, cervical cancer and chronic fatigue syndrome. Dr. Vernon has initiated and participated in numerous international and multidisciplinary research collaborations and she now leads the CFIDS Association’s research program. The CFIDS Association of America is the nation’s largest philanthropic supporters of CFS research.

IIt is interesting that the studies mentioned were not really replication attempts but more likely detractors attempting to validate WPI's brilliant work. It is notable that neither of the two studies found XMRV in the control (healthy) group, which would they would be expected to find at about 3 - 4 %. This suggests that their testing methods were faulty. It is also interesting that the first group of 500 patients in Britain being tested for XMRV by WPI have returned a whopping 95% positive result. A further study of 500 more British patients is about to start. I have great hopes that this will lead to treatments and a better understanding of ME/CFS. My one fear is that XMRV will attract the same kind of discrimination as HIV in its early days.

Latest news: ‘UMC St. Radboud’s’ blood XMRV-positive after all

Gendringen, the Netherlands – April 16th, 2010.

Researchers from the UMC St. Radboud announced in February that they didn’t find the XMRV virus in the blood of Dutch Chronic Fatigue Syndrome (ME/CFS) patients. However, they left out that American researchers did find the XMRV virus in blood samples, taken from the same patients.

This is concluded from a letter that Annette Whittemore, director from the Whittemore-Peterson Institute (WPI) in Reno, sent on Monday, April 12, to Dr. Myra McClure (1). Dr. McClure is a professor in retrovirology at the Imperial College, London, and one of the authors of the first negative “replication study” for XMRV contamination with ME/CFS (2). In her letter, Whittemore invited McClure to co-operate with the WPI to research XMRV. She also reveals some information on the other two negative replication studies that have been produced so far, including the UMC St. Radboud study.

Email correspondence

The experimental virologist Dr. Frank van Kuppeveld from UMC St. Radboud and internist doctor Jos van der Meer didn’t find a trace of XMRV in the frozen blood of 32 Dutch CFS patients, taken in 1991 and 1992. Also, in the blood of 43 healthy control subjects they didn’t find the retrovirus. They published their findings online in the British Medical Journal (3), late January.

However, in her letter, Whittemore points out that the WPI, at the request of van Kuppeveld, has tested some blood samples from the Dutch research cohort before the study at UMC St. Radboud was
completed. The WPI found traces of XMRV in those blood samples. Whittemore claims she possesses over email correspondence, which proves that van Kuppeveld was informed about these WPI research results before he published his negative study. However, in his scientific publication, no word is spoken about the co-operation with WPI (3). The redaction at Ortho has requested a copy of the email correspondence with UMC St. Radboud from Annette Whittemore, but this request has not (yet) been honoured.

Furthermore, Whittemore writes in her letter that van Kuppeveld has asked WPI for the reagents and a positive blood sample to determine if his test procedure was able to detect XMRV in positive blood.
The WPI met that request. In her letter, Annette Whittemore questions why he didn’t use these materials in his research.

Confirmation

Annette Whittemore’s letter raises a lot of questions concerning the UMC St. Radboud XMRV study. The most pressing question is whether Van Kuppeveld can confirm WPI’s findings. And if so, how many blood samples were sent from the Dutch research cohort to Reno, and how many positive samples did WPI find? And last, but not least, why did the UMC St. Radboud researchers keep silent about all of this in and around their research publication in the British Medical Journal?

Last Wednesday, the redaction of Ortho called virologist van Kuppeveld after emailing him Annette Whittemore’s letter. During the call, he declared he had never seen that letter before. Van Kuppeveld also said he wasn’t able to respond on such short notice. “I will first have to study the letter, and at least discuss this with my colleagues, who might be out of the country right now,” he said.

However, after some urging, van Kuppeveld confirmed Whittemore’s findings. He says that the case is slightly more complicated then the letter assumes. According to him, one or more of the 43 healthy control subjects from the UMC St. Radboud cohort has been positively tested on XMRV by the WPI. In conjunction with the findings of van Kuppeveld, this has raised some questions concerning the reliability of the WPI methods. Besides this, he has also stated that he had requested more samples from WPI, but has never received more than one XMRV positive sample.

Seven blood samples

Searching for answers, we contacted Dr. Judy Mikovits yesterday. She is the research director for WPI and leader of the XMRV research. She gave us several answers during a telephone interview. “Frank Van Kuppeveld has sent us seven samples”, Mikovits said. “They were numbered 1 to 7. It was about cDNA, that he had made out of RNA”.

WPI tested these seven samples with advanced PCR techniques in a closed system, so that contamination was impossible. Three samples appeared to be positive. After they reported the positive numbers to UMC St. Radboud, a message was returned, saying it concerned 2 patients and one control subject. For Mikovits, this result was expected. “We never were informed how many control persons there were on those seven samples, but two positives in seven is approximately what I expected. I didn’t count on a 100% score, especially not with PCR.

That one control subject has been found positive by WPI was no surprise to Mikovits. “It totally depends on where you get the blood.” At BMJ the UMC St. Radboud researchers have declared that the control samples were taken from people from the same environment as the patients. “The positive control subject is no surprise if it concerns family or a care taker. Control subjects that come with the patients to give blood we call contact-controls. Some of these people might be infected.”

It is a fact that XMRV has been discovered with healthy persons. With WPI’s own Science Research, in a group of 218 healthy control persons, showed that 8 people had a positive XMRV test (3.7%) (4).

No replication

Judy Mikovits and her colleagues are disappointed that the UMC St. Radboud research group has stated nothing about the co-operation with WPI in the BMJ publication. “During a month, we contacted each other at least every 3 or 4 days,’ she said. “Material went back and forth, including co-operation agreements, signatures.” She felt van Kuppeveld was keeping her on her toes. “He was very strenuous and kept asking whether I had received, tested or looked at stuff.”

About a week before the BMJ published the UMC St. Radboud online, the communication fell silent. “That was after I had sent the positive results,” said Mikovits. “I considered it to be good news. You’ve got something to work with. But Van Kuppeveld didn’t consider it good news, because they didn’t find anything. His message was, on your side, there must have been contamination. Even though I got his material, I was speechless. That was the end of our contact. A week later their publication followed.”

Mikovits finds it unbelievable that the UMC St. Radboud researchers haven’t used the material they got from WPI in any way. They did claim to have done a replication study. “We sent them antibodies, positive serum and positive DNA,” said Mikovits. “Van Kuppeveld could have cultivated his samples, just like we did in our Science study. They could have tested their plasma for antibodies and they could have used our reagents to search for proteins and that kind of stuff. But they didn’t, and have also mentioned nothing about the possibilities to do it. We would have wanted Van Kuppeveld to report all the data. If there is a difference in opinion or a misinterpretation, you can look at it together. They could have adjourned the samples, and worked together with us. But you can’t just create the appearance to the outside world that nothing happened.”

In retrospect, van Kuppeveld and his colleagues only were interested in the PCR technique, while Mikovits assumed that at the UMC St. Radboud the entire Science study would be replicated. “I had no idea he didn’t want to do the rest of the research. That totally surprised me.“

Silence

The WPI kept silent until the beginning of this week. Mikovits suspects that Annette Whittemore was triggered by the statements Dr. Myra McClure recently made on television, where the British retrovirologist disregarded the meaning of XMRV with ME/CFS. After this, Whittemore decided to enter the arena with this letter. “For a long time we thought the best way to deal with this was to continue with the research and forget about the lies. What else can you do? We got seven samples, we did our jobs and reported honestly what we found.”

Mikovits confirmed that van Kuppeveld requested more samples. “I wanted to send him more, but what would he have done with it? Find out they were negative and say bad things about it? Mikovits is determined to walk the XMRV-route further, and she sounds more motivated than ever. “We’ve isolated a virus, and we displayed from a hundred people how their immune system is reacting. Did you know that there is no immune response to a contaminant? The patients obviously are infected with a virus.”

Mikovits isn’t the only one that takes XMRV seriously. Previously this month it was announced that in Canada people with ME/CFS aren’t allowed to donate blood. Canada is the first country in the world that has taken this precaution.

1. Whittemore A, Dear Dr. McClure, April 12th 2010 ( http://www.wpinstitute.org)
2. Erlwein ), Kaye S, [..], Cleare A. Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome. PLoS ONE 2010; 1e8519
3. Van Kuppeveld FJ, de Jong AS, [..] van der Meer JWM. 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
4. Lombardi VC, Ruscetti FW, [..], Mikovits JA. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 2009; 326(5952):585-9

Source: http://www.mecvs.net/module-ME_CVS_docs-viewpub-tid-1-pid-537.html