Second XMRV Negative Study …
Still In Search of a Proper and Robust Replication Study
Suzanne D. Vernon, PhD
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.
This study included three different cohorts:
- 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.
- 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.
- 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 SciencesRetrovirologystudy. The high rates of XMRV antibodies described in the Science 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 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.
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.