This is very long, however, I could not get the webaddress so people could cut and paste. gap 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/presentations/xmrv_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.