By Suzanne Vernon, PhD, Scientific Director
The 2009 Science paper by Lombardi et al. was a seminal investigation that mobilized many in virology, blood safety and the media to understand XMRV and determine its association with CFS. In today’s issue of Science, a partial retraction of the 2009 Lombardi et al. paper is published with Robert H. Silverman of the Cleveland Clinic as the lead author. This issue of Science also features the long-awaited results of the Blood XMRV Scientific Research Working Group (SRWG) in a paper titled, “Failure to confirm XMRV/MLVs in the blood of patients with CFS: a multi-laboratory study” with Graham Simmons of Blood Systems Research Institute and University of California, San Francisco as the lead author. I’ll break down these papers in two parts.
Part 1: The Partial Retraction
An eight-page feature story titled “False Positive” accompanies these papers and chronicles the events that led to the publication of the 2009 Science paper and backgrounds of many of the key players. Silverman, who discovered XMRV, collaborated with the Whittemore Peterson Institute (WPI) team and provided them with XMRV reagents so they could pursue what seemed like a promising avenue of CFS research. With repeated failures to replicate the initial CFS/XMRV report, the incongruent results reported from Phase II of the SRWG and the possibility that XMRV originated from passage through laboratory mice, Silverman and his colleague Jaydip Das Gupta set out to reexamine CFS and healthy control DNA samples originally received from the WPI.
Structure of XMRV, courtesy of Nature Reviews Urology
Using immaculate laboratory and PCR procedures, Silverman and Das Gupta detected the XMRV VP62 plasmid in 7 of the 15 CFS patient DNA samples and 0 of 17 healthy control DNA samples. This XMRV VP62 plasmid is a full-length XMRV molecular viral clone that can produce infectious XMRV virus when transfected into LNCaP cells. Plasmids can be thought of as DNA transfer vehicles, containing a few other genes that are not XMRV-specific but help the plasmid in producing high levels of infectious virus. Silverman and Das Gupta first used single round PCR for the XMRV env gene and detected this XMRV gene in 6 of the 15 CFS patient DNA samples and 0 of 17 healthy controls. The six CFS XMRV env positive samples were also positive for a piece of the VP62 plasmid DNA specific to the plasmid and not XMRV, called neo. None of the healthy control DNA samples had neo plasmid DNA amplified. Further confirmation of plasmid contamination was demonstrated by single round PCR of the CMV promoter region of the plasmid in the same 6 CFS DNA samples and none of the controls. They amplified and sequenced DNA from another CFS sample identified as WPI-1124. This step verified that seven of the CFS patient samples received from the WPI and shown in Figure 1 of the 2009 Science paper were positive for the XMRV VP62 plasmid. DNA sequence and analysis from the positive CFS patient samples also indicated that data in Table S1 and Figure S2 of the Science 2009 paper was likely “spurious and due to contamination with the XMRV VP62 plasmid DNA,” the retraction states.
This work by Silverman and Das Gupta indicates the source of XMRV in the CFS patient samples as the original full-length XMRV plasmid rather than naturally occurring XMRV. They reached this conclusion based on the fact that they easily amplified portions of the plasmid DNA that is not naturally occurring DNA. This partial retraction of data from the Science 2009 paper, signed by all of the authors on the original paper, casts further doubt on the validity of the original report and the likelihood that naturally occurring XMRV is associated with CFS. The editors of Science have indicated that they are discussing “next steps” with the authors and that they stand by the Expression of Editorial Concern published in May.
Part 2: The Blood Safety Study
[Note: Study authors provided a webinar presentation about this study for the Association. Recording, slides and more info: http://www.research1st.com/2011/10/14/xmrv-updates/]
In the Science 2009 paper, Lombardi et al. stated that XMRV could be detected in the blood of 8 of 218 healthy controls. This raised the possibility that XMRV could be a threat to the blood supply. This type of threat is not new to the community of scientists who deal daily with maintaining a safe blood supply and within a month, the Blood XMRV Scientific Research Working Group (SRWG) was formed. Its primary purpose was to design and carry out a four-phase study to determine whether XMRV posed a threat to blood safety. An overview of this four-phase study was published in March 2011 in Transfusion. Phase I and II focused on assay development, optimization and standardization. The aim of Phase III was to develop coded panels of clinical samples from CFS patients who had tested positive for XMRV in the 2009 Science paper and P-MLVs as described in the 2010 paper by Lo et al., pedigreed negative controls (samples tested repeatedly and consistently shown by all participating laboratories to be negative for XMRV and P-MLVs) and experimentally prepared positive controls also known as “spiked” controls. Phase IV would use optimized assays and conditions to test for XMRV and P-MLVs in a large number of samples collected from healthy blood donors.
The paper published today in Science titled, “Failure to confirm XMRV/MLVs in the blood of patients with chronic fatigue syndrome: A multi-laboratory study,” by Simmons et al., finishes the work of the SRWG. Nine independent laboratories (see figure at right) using 19 optimized and highly sensitive assays were unable to reproducibly detect XMRV or MLVs in the coded panel of samples. The coded panel consisted of blood samples from five CFS patients who were positive for P-MLVs as described in the Lo et al. paper and 10 CFS patients recruited by the WPI who had previously tested positive for XMRV by at least one of a variety of test methods. Samples from six of these CFS patients were also used in the original study published in Science in 2009. The study design as described in Transfusion called for a larger panel of 30 previously-positive CFS subjects; however, Simmons et al., note that this smaller group was, “the maximum number of subjects who could be recruited by the cohort investigators.”
As noted above, the coded panel included pedigreed negative controls collected from three laboratory personnel and 12 local blood donors from Blood Centers of the Pacific, San Francisco. These negative control blood samples were processed into various blood components needed for the assays and distributed to participating laboratories for XMRV/MLV. All participating laboratories were in unanimous agreement that these controls were pedigreed negative. Finally, three experimental positive controls were prepared by spiking samples with cells or viral RNA from 22Rv1 cells to serve as an XMRV positive controls. All samples were coded and included as duplicates or triplicates in the panel.
Samples were collected, processed and prepared under stringently controlled, identical procedures. Sources of contamination of supplies and materials were carefully eliminated. All of the samples were coded and tested under blinded conditions, meaning that the laboratory personal doing the tests did not know whether they were testing samples from CFS patients, blood donors, pedigreed negatives or spiked positive controls. Each of the laboratories was able to define the assays it used to test samples. In total, there were 19 different assays used that fell into three categories: polymerase chain reaction (PCR), antibody reactivity and culture. The WPI has stated that culture is its most sensitive means of detecting XMRV and other gammaretroviruses; however, a laboratory problem with mycoplasma contamination prevented it from testing samples using this method. Dr. Francis Ruscetti’s lab at the National Cancer Institute (NCI) and Dr. Indira Hewlett’s lab at the Food and Drug Administration (FDA) used methods identical to WPI, as described in the supporting materials for the study.
Phase III results: red indicates positive results; orange reflects indeterminate results
The labs completed their work, submitted results and the code was broken by staff at the Blood Systems Research Institute in August. Only two of the nine laboratories, the WPI and Dr. Francis W. Ruscetti’s laboratory at the NCI, detected XMRV/MLV in any of the clinical samples contained in the coded panel. The remaining seven laboratories, including Dr. Lo’s FDA lab, only detected XMRV/MLV in the spiked positive controls, each with high accuracy. Only the WPI detected XMRV/MLVs in the CFS samples using PCR assays. The WPI assays were the least sensitive for detecting viral RNA as illustrated by the inability to detect RNA in 2 of 5 spiked plasma samples and 1 of 5 PBMC-spiked samples. The WPI and NCI/Ruscetti laboratories detected XMRV/MLVs at the same rate in CFS patient samples as in healthy blood donor samples. Further, even though each sample was represented in duplicate or triplicate, WPI and NCI/Ruscetti frequently only detected XMRV/MLVs in one of the duplicate or triplicate samples from the same subject. Sequence analysis of DNA amplified from the positive assays showed a high degree of similarity to XMRV derived from 22Rv1. Mouse DNA contamination was not found in any of the positive samples. Using the kappa coefficient as a statistical measure of agreement, it was found that even though both WPI and NCI/Ruscetti detected XMRV/MLV in some of the samples, the samples that were positive were often different samples, indicating there was no agreement on what could be called an XMRV/MLV positive sample.
The conclusion of this nearly two-year study is that XMRV/P-MLVs are not reproducibly detected in clinical samples (15 from individuals who had previously tested positive) using current, state-of-the art assays. The authors state, “our findings are reassuring with respect to blood safety and indicate that routine blood donor screening for XMRV/P-MLV is not warranted at this time.” Phase IV will not proceed, although there are results from studies of large numbers of blood donor samples pending publication.
With this closing statement, it appears that the blood safety community is satisfied with the scientific rigor of the SRWG study and that XMRV/P-MLVs are not a threat to the blood supply. It is remarkable to see this type of study done with government, commercial and private laboratories working in parallel to address a potentially serious public health issue. Even though the SRWG studied was designed as a blood safety study, it helped bring CFS into the light. Many of investigators on the SRWG previously knew little about CFS. Now they are familiar with the enormous suffering that CFS inflicts, the problems with the CFS case definition for research and the anger and passion that comes with an illness that has so long been in the dark shadows, unsolved.
Suzanne Vernon, PhD, is the Association’s scientific director. She has nearly two decades of experience as a microbiologist.
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“All publicity is good publicity.” Alan Sorensen, an Econ professor at Stanford, looked at NYT book reviews. He determined that, even when reviews were negative, previously unknown authors saw a significant bump in sales. This publicity in the journal Science is good publicity. It is a tacit acknowledgment that the research goals are legitimate and that CFS is a real non-psychiatric disorder.
It’s heartening to see the medical research community finally getting the message about CFS. My wife of 40 years suffered from increasingly severe symptoms of CFS for nearly a decade before finally being ‘officially’ diagnosed with the disease in 2010. She died of a host of CFS-related complications on August 2, 2011 in a leading San Francisco Bay Area hospital, after being in ICU for 8 days during which all the drips and pills they could throw at her did no more than relieve just some of the pain she was suffering. It goes without saying that I’m grateful that they used what they knew to try to help her. The problem, of course, is that — in spite of my near-constant attempted explanations — virtually no one in this excellent ICU facility had the slightest understanding of what they were dealing with or how to treat it. I very much hope this will change before too long, and before too much more suffering by others.
I’m so sorry for your loss. I have fibromyalgia 20 years and the past 4 years have been diagnosed with chronic fatigue. This is worse than the 20 years of pain. It seems like everytime you turn around something else is going on with the body. We live from hour to hour it seems. It is a roller coaster ride of life and thank God for the good days. If you are up to answering me I am really interested in knowing what was your wife’s complications that she had to deal with, no one believes this disease actually can lead to death. Once again I’m so very sorry. My husband is a saint to me and that YOU were a blessing to your wife. 40 years, how sad that this disease took her. Prayers for you and her.
Interesting. I got CFIDS in 1989, diagnosed in 1990 . I did not get Fibro until after a neck surgery in 1995.
Just in time ! LOL
Glad they are keeping up the research .A little late for me, now that I’m 60
Hi Kathy, I don’t know if you follow FM research too, but you might want to check into the possibility that your neck surgery triggered FM as the result of a compression of your spinal cord. Here is a link to the National FM Research Association that invested heavily in research linking FM to Chiari malformation and cervical stenosis. http://www.nfra.net/NeuroCon.htm It remains a somewhat controversial area of research and treatment. There have been successes and disappointments reported by patients with FM, CFS and both diagnoses who have had the surgery.
Kim ,
Yes, Over the years with all the MRIs of my neck, my cerebellar tonsils are lower into the upper cervical spinal canal. There is aqcquired Chiari Malformation. Probably from my neck injury. Its not dropped enough to diagnose it. I read it on my MRI report.
This XMRV study is interesting, and I wonder if the “synthetic” XMRV would test positive. Probably not.
(Surprisingly it was neither identical to MuLV nor to the novel xenotropic MuLV related retrovirus (XMRV) but showed 99% identity to a synthetic retrovirus which was engineered in the 1980s.)
Link :http://www.ncbi.nlm.nih.gov/pubmed/19772602
I am surprised they have not deleted this page….Just sayin …
It was in the 80′s that people started getting sick . All my antibodies were positive in 1990 that they looked for in order to diagnose it. Epstein Bar 1:1280,ANA, EBEA,EBNA,HHV6,Low Supressor T-cell,High Killer T-cell,(shows overactive immune),and IGG, anti-thyrogloblin….and more I forgot without getting the report.
I have kept up with all this for years, but have really run out of hope for getting my life back now. Its just too late, and too tired .
Kathy
The paper you cite got a lot of attention several months back. Zhang et al. also report wide spread of XMRV contamination in cell lines used in research: http://www.landesbioscience.com/journals/cbt/article/15955/. It’s important to distinguish that that there is diminishing support for the hypothesis that these viruses infect humans and cause disease, even if they are a problem for labs that use these cell lines. A study published this week shows that XMRV was not present in cell lines used for vaccine development and production. Investigation of XMRV in human and other cell lines, Williams DK, Galvin TA, Ma H, Khan AF., Biologicals, Oct. 12, 2011
http://www.sciencedirect.com/science/article/pii/S1045105611001205.
There is a great deal of historical evidence to show that CFS-like illnesses exist by other names throughout history. The medical literature of the 20th century contains many descriptions using various names of the same set of symptoms that are now identified as CFS or ME or ME/CFS. Here’s one source of more information about that topic: http://www.healthcentral.com/chronic-pain/chronic-fatigue-151675-5.html
There is more attention and more research being focused on CFS than ever before. The tools available to researchers to look inside the body, its systems and components are enabling new insights to guide objective diagnosis and focused treatment. Please hang in there.
Dear Mr. Brown, I cannot tell you how sorry I am. May I offer my sympathy to you & your family.
BUT that is not the only reason I am emailing you. May I ask Please, what was the reason your wife died???
I was diagnosed with Fibromyalgia at 22 & CFS fallowed closely, more & more. NOW my entire live is bent around CFS & the other complications that come with it.
Just a side point, the rest of the world called it CFIDS, “Chronic Fatigue & Immune Dysfunction Syndrome,” I believe it is more accurate.
My Fav scripture is Revelations 21:1-5, I hope you to may find some comfort in it!
May the future hold comfort & peace.
I am very sorry I forgot, which is so easy these day, I’ve had CFS for 35 yrs.
I send my deepest regrets to you on the loss of your wife.
My daughter has suffered from cfs since 1999 at age 15 it has stolen her life although she has shown improvement everyday is a struggle. May you find peace.
So are we, the people with CFIDS/ME able to donate blood again, and organs? Or are we still prohibited from donating blood? My Doctor has asked me this question………..he wants to learn about CFIDS, thank you, Dr Foley
Teresa, your question is very pertinent. Because the SRWG study was organized as a means to evaluate the risk of XMRV to the blood supply, its conclusions will be of interest to blood collection organizations in the U.S. and other countries. The CFIDS Association restates its long-standing recommendation that individuals with CFS not donate blood or organs. (see http://www.cfids.org/blood.asp) The AABB Interorganizational Task Force on XMRV issued a statement today (http://www.aabb.org/pressroom/statements/Pages/statement092211.aspx) that blood screening for XMRV is not indicated by the available data and that it will re-evaluate blood donation guidelines as they apply to people who have had CFS in the past but now feel fully recovered. The prudent thing to do, for the safety of donor and recipient, is if you have CFS, please don’t donate blood. For more info on this topics, please see “XMRV and Science: Take Three” at http://www.research1st.com/2011/09/22/science-xmrv/.
It makes logical sense for people with CFS/ME not to donate blood, because even if the disease is not caused by XMRV/MLV viruses, something physiological IS causing it. And if it is a contagious agent, why subject anyone else to it — especially someone who is ill or weak, which would make them vulnerable to another disease.
More information is being found by studies — Komaroff, Klimas, Light, Natelson, etc. that explains organic factors; something is causing the changes in brain matter, genetic expression, spinal fluid proteins, etc. We hope the research will proceed quickly and that conclusions will be reached which can help us — and help doctors, researchers, family, friends, the wider community, insurance companies (well, those may be hopeless, as they don’t want to pay more claims) understand this disease and figure out treatments.
Is there any evidence (or has anyone heard of any stories) about a family member “catching” CFS from a loved one? If it is indeed a virus, is it one that could spread from a spouse to spouse? Has anyone ever heard of this happening? I haven’t. But, I don’t have my ears to the ground as much as others.
Dear CJ,
I have CFS since 1991 and am now 54, contracted it after an Influenza A illness. A few months later I married, my husband was healthy. He contracted CFS after about 4 years and is worse than me. Although a well-known MD/professor researching on CFS at that time thought CFS couldn’t be given to someone else, I am 100% convinced he got it from me! And I pray that my two daughters will not get the disease, but one of the two (they are twins) is often tired!
Fortunately we have a lighter form of CFS, we can still go to work but are exhausted at the end of the day and in the weekend. And of course lots of symptoms, mood changes, nerves, insomnia, irritatable bowel syndrome, arthritis, etc etc….
So my idea is that CFS is sexually transmissible, and think that giving blood is therefore not a thing to do.
My wife did get Fibro-Myalgia 10 years ago and I contracted CFS 4 years ago, bveing still on disability: is there a link? I do NOT know, but there have been epidemics of CFS…
What seems to me very interesting is the fact of “Persistent Human Leptospirosis” (www.leptospirosis.org/topic.php?t=41) where some bacteria can persist in “I.P” or “Immuno-Priviliged sites”, in fact the ocular fluids, central nervous system and kidneys tubules, i.e. sites where the immune system is poorly active…and where bacteria can be “trapped” and somehow lead to some pathogenic processes! Another clue to this possibility could be the occurence of “Numerous studies demonstrating a HIGH incidence of CHRONIC INFECTIONS in CFS and FM”: article published by Dr.Holtorf on May 2011,and insisting on the absence of elevation of IgM and IgG in these cases !(www.ProHealth.com). So…what if some germs, viruses or other pathogens, from time to time, emerge from these “I.P.” sites and infect temporarily the blood stream???? and what about a study which studies ONLY 14 patients and their blood ONLY at ONE precise moment?
Mr Brown, I am so sorry for the loss of your wife and I certainly understand the suffering of CFIDS/CFS/Fibro because of my almost 30 years of suffering 24-7 with this disease and now at age 67 living with the progression of it which has caused numerous symptoms. I wish you would share a little more about what happened to your wife because I just don’t understand how she could have been diagnosed in 2010 and got so sick that she died by 2011. This disease is so destructive to the human body, however it is slow moving and keeps destroying a life in many ways. Then to make matters worse it is not taken seriously by friends, family and most of the scientific community.
I will continue to fight to keep my so called life and just live in hope of a better future for all of us that suffer with this devastating disease.