http://www.cfids.org/cfidslink/2010/010603.asp XMRV Negative Results Emphasize Need for Robust Replication Study Suzanne D. Vernon, PhD, Scientific Director A study testing for evidence of XMRV infection in CFS patients in the United Kingdom has reported negative results. This is the first publication following the article in the top-ranked journal Science from researchers at the Whittemore Peterson Institute, the National Cancer Institute and Cleveland Clinic that garnered worldwide attention from the media and scientific community. The new report, published Jan. 6, 2010, in the open access online journal PLoS ONE, failed to detect XMRV in CFS, but should not be considered a valid attempt to replicate the findings described by Lombardi et al., in the Oct. 8, 2009 Science article. The PLoS ONE paper by Otto Erlwein, Steve Kaye, Myra O. McClure, Jonathan Weber, Gillian Wills, David Collier, Simon Wessely and Anthony Cleare is titled, “Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome.” The investigators tested peripheral blood DNA from 186 routine clinic attendees who met 1994 (Fukuda) CFS case definition criteria and were well-characterized from participation in prior neuroendocrine and cognitive behavioral therapy studies. These 186 CFS patients were reported to be unwell for a median of four years with high levels of fatigue and disability. This team of researchers used a special type of DNA “xeroxing” called nested polymerase chain reaction (PCR) reaction to amplify specific segments of the XMRV proviral DNA from the genomic DNA obtained from these 186 CFS subjects. In essence, they were looking to see if XMRV genetic material had integrated into human genetic material, which is a key characteristic of retroviral infection. The experiment included positive, negative and contamination controls, but did not test any samples taken from healthy subjects. The samples were coded so that the origin of the DNA was not known to the person conducting the PCR assays. XMRV was not detected in any of the 186 samples. Can this study be considered comparable to the results published by Lombardi et al., in Science? In short, no. Both studies included CFS patients defined by the 1994 case definition criteria, but this is where the comparability ends. Here are some of the ways the PLoS ONE and Science methods differ: The blood was collected from CFS patients in different types of blood collection tubes. The genomic DNA was extracted and purified using different techniques. The amount of genomic DNA included in the amplification assay was different. Different primer sequences were used that amplified different regions of the XMRV proviral DNA. The conditions of the PCR amplification assay were different – from the numbers of cycles, to the type of polymerase used. Should these differences affect an investigator’s ability to detect XMRV? To a microbiologist with experience handling samples and studying various infectious agents (as I am), these variances in procedure could make the difference between detecting XMRV or not. It very well could be true that XMRV is not present in the U.K. as Erlwein, et al. suggest in their discussion, but it is also possible that the technique used in the PLoS ONE paper was suboptimal due to the different methods employed, when compared to the original experiments conducted by Lombardi, et al. The U.S. Department of Health and Human Services Blood XMRV Scientific Research Working Group has been established to delineate the research studies that should be undertaken to evaluate whether XMRV represents a risk to the safety of the blood supply. As a first step in this evaluation, analytical panels are being developed by the National Heart, Lung, and Blood Institute Retrovirus Epidemiology Donor Study-II (REDS-II) that will allow for 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. We look forward to the results of this study and urge that it be completed expeditiously, especially in light of this report from the U.K. In the meantime, be prepared to read about more studies with conflicting findings. Rather than simply accept or dismiss new information, we will help make sense of why discrepant results occur. Perhaps the most important statement in the PLoS ONE paper is the acknowledgement by this group of investigators that CFS is an incapacitating organic disease affecting millions of people worldwide. Once XMRV detection methods are optimized and made widely available, we encourage this group of researchers to take another look at XMRV as a possible explanation for the organic basis of CFS in the U.K. Citations: 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 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. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 8 October 2009. 1179052.