A Twitter tweet from the June 1 London 'Invest in ME Conference' has Dr. Daniel Peterson announcing that the Lipkin study is completed, with an expected release date of June 30, 2012. Virus hunter Ian Lipkin, MD, at Columbia has been heading up what he calls "the XMRV/MLV/CFS/ME study" to search for evidence of viruses in blood samples from a well-characterized, geographically-distributed US cohort of ME/CFS patients, using cutting edge virus-detecting technology.
We'll keep you posted!
Lo/Alter Retract pMLV Finding in CFS
by CORT on DECEMBER 27, 2011. Edited by ME/CFS Assist for grammar and syntax only.
Just fours days after the editors of Science took it upon themselves to retract the 2008 Science paper, Lo/Alter, the authors of the FDA study at [who at] one time championed as proof that a family of XMRV-like viruses was present in ME/CFS, have retracted their PNAS findings as well.
In their conclusion to be published next week they stated:
"Although a more definitive, National Institute of Allergy and Infectious Diseases (NIAID)–sponsored, coded panel of samples from 150 well-characterized and geographically diverse CFS patients and controls is being assembled for further study, in consideration of the aggregate data from our own laboratory and that of others, it is our current view that the association of murine gamma retroviruses with CFS has not withstood the test of time or of independent verification and that this association is now tenuous. Therefore, we retract the conclusions in our article.”
The Alter/Lo retraction deepens one conundrum as they reported that extensive tests for contamination tests still failed to find any. Their retraction rested on:
Earlier this week Lipkin gave his vote of no-confidence in the original XMRV finding stating that he felt the only reason left for the 2.3 million NAIAD study to go forward was the possibility that Dr. Mikovit had found other closely related viruses. The Lo/Alter retraction may increase calls for the Lipkin study to be pared back. Raccaniello simply stated that the Lipkin study seemed ‘less compelling’ in light of the recent findings and questioned whether in light of the ‘many negative’ studies one more study will make any difference.
- Their inability to find the MLV’s in the same patients they found them in before (as well as other CFS patients) in the Blood Working Group study
- The inability of numerous other laboratories to find evidence of MLV’s (6 labs in the BWG study and others in published studies)
- Their and other labs inability to find anti-XMRV antibodies, XMRV virions, or viral integration sites in patient samples that would confirm their original findings.
- The lack of a match between the MLV’s they found in stored samples and in the same patients over a decade later. (This was known at the time of publication but for some reason was not released. Reports at the time suggested that the present day MLV’s sequences were descendants of the earlier MLV’s found but this was not true.)
- Their inability to give any more of the original samples to other labs.
(Lipkin’s scenario seems a bit tenuous. The idea that the WPI researchers had found a contaminant (XMRV) that just happened to look like another retrovirus (MLVs) that was present seems highly improbable; the equivalent of a researcher throwing a dart into a milky way of genetic sequences and just happening to land on the right one. If XMRV was present in CFS patients then the idea of similar ‘HGRVs’ made sense; if it wasn’t (ie if it was just a contaminant) then it beggared the mind to think the WPI would, by happenstance, happen to find another retrovirus that looked similar to it.)
Agonizingly Powerful Technology - If anything the XMRV/pMLV saga underscores a technology (PCR) whose sensitivity has, to some extent, outrun researchers ability to easily interpret it. Hanson’s report that she could find the virus in one PCR machine but not the other and Singh’s conclusion that a supposedly clean machine harbored XMRV-like sequences underscored how difficult it is to interpret positive results in this field. Both researchers undertook painstaking and time-consuming re-examinations of their work before they were able to conclude their results were due to contamination.
Despite calls from colleagues to do the same the WPI does not appear to have investigated the issue rigorously until Silverman found evidence of contamination in his samples. At that point the WPI used an independent lab to determine whether contamination was present in their samples; that lab found contamination in at least some of the WPI’s samples.
Last Chance for XMRV – Dr. Mikovits and Dr. Ruscetti are in the odd situation of agreeing with all the findings; they accept that contamination was present in the original study and also believe that XMRV as well as XMRV-like sequences are present as well. Dr. Mikovits will, with Dr. Ruscetti, be looking for ‘XMRV’ in the Lipkin NAIAD study due to wrap up, David Tuller of the New York Times reported, in March of next year.
Researcher Confesses: Implicates Dr. Mikovits in Theft of Materials from Whittemore Peterson Institute ("WPI")
by CORT on NOVEMBER 22, 2011
Dr. Judy Mikovits has had a difficult couple of months. First the BWG study indicated the tests she developed for XMRV were not valid, then evidence of contamination resulted in a partial retraction of the original Science paper, then she was accused of doctoring a photo at a conference and in the original Science paper prompting several investigation, then she was terminated from her job at the WPI, then she was served with a lawsuit claiming that she had stolen data from the WPI and since last week, she’s being in the Ventura County jail waiting arraignment as a fugitive from justice because, against court orders, she crossed the Nevada border into California. She must be wondering if it could get any worse. It has - but first some background.
The latest episode began with a lawsuit filed by the WPI a month after Dr. Mikovits termination claiming that Dr. Mikovits had taken critical notebooks and flash drives from the WPI after she was fired. According to ScienceInsider Dr Mikovits had the only key to the desk the notebooks were kept in. After Dr. Mikovits termination the WPI had the desk manufacturer open the desk for them and found it empty of her and other researchers notebooks. A source at the WPI said that it was her understanding that Dr. Mikovits desk had been ‘emptied out’ and up to five years research data was gone. They then served notice of the theft to the police and a police investigation began.
Why are these laboratory notebooks so important? Because they serve as the document of record in scientific research and are specially manufactured and used in specific ways…..
WIKIPEDIA – A lab notebook is a primary record of research…..To ensure that the data cannot be easily altered, notebooks with permanently bound and numbered pages are often recommended. Researchers are often encouraged to write only with unereasable pen, to sign and date each page, and to have their notebooks inspected periodically by another scientist who can read and understand it. All of these guidelines can be useful in proving exactly when a discovery was made, in the case of a patent dispute.
After several failed attempts to get the materials back the WPI filed a lawsuit a month later to compel Dr. Mikovits to return notebooks, flash drives, information on her laptop computer and information in her email account. In a blog titled “Wings of Hope: Our Responsibility” the WPI stated that the failed efforts to get the materials back forced had forced them to engage in ‘costly litigation”. The data loss included 12-20 notebooks dating back five years. The WPI stated that, "the costs of legal action pale in comparison to the years of expenses incurred for researcher salaries, equipment, and supplies to generate this valuable work”.
(Dr. Mikovits contract stipulated that all intellectual property she produced while under the employ of the WPI was the property of the WPI.) The WPI also won a temporary restraining order stopping Dr. Mikovits from “destroying, deleting or altering’ any of the materials that may be in her position.
For her part, through her attorney Dr. Mikovits stated that she received notice of her firing on her cell phone and then immediately left for her home in Southern California without visiting the Institute again. She stated she had placed her keys in unlocked drawers in her lab as was her custom so that other lab personnel would have access to the notebooks. Her attorney stated that:
Dr. Mikovits was not and is not in possession of the lab notebooks or any WPI intellectual property. A number of individuals have keys to the office and lab, including the administrative staff, lab staff and custodial.
Instead of Dr. Mikovits returning the materials her attorney requested that her notebooks be returned to her so she can continue to work on the grants she won while employed at the WPI. Her attorney stated that:
"Dr. Mikovits’ notebooks, as well as those of the employees whom she supervised, should be returned to Dr. Mikovits so she can fulfill her responsibilities as PI on these government grants and corporate contacts".
The WPI lawsuit does not request just the return of the materials; if found guilty it also requests that Dr. Mikovits be liable for attorneys fees, punitive damages and other damages to be proven at trial – potentially leaving Dr. Mikovits with a substantial financial burden if the WPI prevails.
The Judge overseeing the case ordered Dr. Mikovits not to leave the state of Nevada until the case was resolved. Dr. Mikovits left Nevada and was reportedly arrested at her home in Ventura County on the 18th on felony charges of fleeing justice. She was not allowed to post bail and is being arraigned today.
Research Assistant Confesses Theft - in a legal filing pertaining to a preliminary injunction against Dr. Judy Mikovits the Whittemore Peterson Institute provided evidence late last night that Dr. Mikovits engineered the theft of the research materials from the WPI lab after her termination.
In an affidavit Max Pfost, a researcher working at the Max Pfost, admitted that he removed the documents/flash drives at Dr. Mikovits behest the morning after her termination. According to Pfost’s affidavit, upon hearing of her termination Dr. Mikovits called him and told him that the WPI would ‘go down’. She then met him in a bar and provided him with the keys he needed to take ‘patient samples’, lab notebooks and other information.
Pfost attempted to gain entry to the WPI that night but was unable to and entered the building the next morning when it was unlocked. Pfost then removed between 12-20 heavy notebooks containing approximately five years of research work from Dr. Mikovits, Pfost and two other researchers. Unable to fit all the notebooks into his backpack Pfost carried others out in his arms to his car and then to his apartment where he hid them in a “Happy Birthday” bag. Realizing that the WPI was searching for them he then moved them to his mothers' house.
Dr. Mikovits requested that he mail them to her at her house but Pfost protested that the size and the weight of the notebooks made them too expensive to mail. After confessing to his mother that he had stolen the notebooks Pfost removed them from her house and returned them to his condominium.
Mikovits In Hiding – Shortly after meeting Dr. Mikovits early in the morning at the Reno airport Pfost handed over the notebooks and other materials to her. Dr. Mikovits informed Pfost that she was hiding out on a boat to avoid being served papers by the WPI and requested that he inform her of the whereabouts of WPI employees to avoid detection.
Attempted Removal of Biological Materials as Well – According to the affadavit Dr. Mikovits also attempted to have biological materials including cell lines and blood samples removed from the labs as well. At Dr. Mikovits behest Pfost attempted to recruit other WPI employees to remove these materials from the lab and send them to Dr. Ruscetti. According to the affidavit research assistant Amanda McKenzie, declined to do so.
The Stolen Materials – A WPI filing asserts that the WPI uses the stolen materials on a ‘daily basis’ and cannot proceed on critical areas of research without it.
WPI needs the Misappropriated Property to research effectively, continue ongoing experiments and studies, communicate with research subjects effectively, apply for patents, recruit researchers, and obtain grants to help find a cure for those patients suffering with NID.
Law Enforcement in Charge – A misunderstanding has arisen about the WPI’s role in Dr. Mikovits arrest. When the WPI realized important documents were missing they reported the theft of the materials to the police. At that point a police investigation was launched; based on the facts of that investigation and the lawsuit filed by the WPI, the Judge overseeing the case produced the initial restraining order stopping Dr. Mikovits from altering or destroying the documents or leaving the state of Nevada. Once Dr. Mikovits left the state of Nevada she was subject to arrest. On Nov 17th a warrant was issued for her arrest and she was arrested last Friday. Dr. Mikovits fate is now in the hands of the legal system. The police will determine whether or not to bring charges.
Felony Theft - When a person steals property, the market value of the property will dictate whether the theft is classified a felony. Under Nevada law, if the value of the stolen property exceeds $250, the crime is a felony. When the value exceeds $2,500, the crime becomes grand theft, punishable by one-to-five years in state prison and up to $10,000 in fines.
If Dr. Mikovits has the documents she certainly has good reason to return them. The WPI’s lawsuit, which was filed over a month ago, asked for legal fees, punitive damages and other damages.
Furthermore Dr. Mikovits has been charged with two felonies; avoiding justice and stealing property from the WPI. In the state of Nevada the market value of the product stolen determines how a theft is classified. Thefts above $250 are considered felonies; thefts above $2,500 are considered ‘grand theft’ and are punishable by 1-5 years in jail and up to $10,000 in fines. According to the affadivit the documents in question represent 5 years of work.
A hearing is scheduled today in Nevada at 1pm PST regarding the preliminary injunction which will expire after tonight.
via the NYT
Carl Zimmer, June 25, 2011
ONE of the great strengths of science is that it can fix its own mistakes. “There are many hypotheses in science which are wrong,” the astrophysicist Carl Sagan once said. “That’s perfectly all right: it’s the aperture to finding out what’s right. Science is a self-correcting process.”
If only it were that simple. Scientists can certainly point with pride to many self-corrections, but science is not like an iPhone; it does not instantly auto-correct. As a series of controversies over the past few months have demonstrated, science fixes its mistakes more slowly, more fitfully and with more difficulty than Sagan’s words would suggest. Science runs forward better than it does backward.
Why? One simple answer is that it takes a lot of time to look back over other scientists’ work and replicate their experiments. Scientists are busy people, scrambling to get grants and tenure. As a result, papers that attract harsh criticism may nonetheless escape the careful scrutiny required if they are to be refuted.
In May, for instance, the journal Science published eight critiques of a controversial paper that it had run in December. In the paper, a team of scientists described a species of bacteria that seemed to defy the known rules of biology by using arsenic instead of phosphorus to build its DNA. Chemists and microbiologists roundly condemned the paper; in the eight critiques, researchers attacked the study for using sloppy techniques and failing to rule out more plausible alternatives.
But none of those critics had actually tried to replicate the initial results. That would take months of research: getting the bacteria from the original team of scientists, rearing them, setting up the experiment, gathering results and interpreting them. Many scientists are leery of spending so much time on what they consider a foregone conclusion, and graduate students are reluctant because they want their first experiments to make a big splash, not confirm what everyone already suspects.
“I’ve got my own science to do,” John Helmann, a microbiologist at Cornell and a critic of the Science paper, told Nature. The most persistent critic, Rosie Redfield, a microbiologist at the University of British Columbia, announced this month on her blog that she would try to replicate the original results — but only the most basic ones, and only for the sake of science’s public reputation. “Scientifically I think trying to replicate the claimed results is a waste of time,” she wrote in an e-mail.
For now, the original paper has not been retracted; the results still stand.
Even when scientists rerun an experiment, and even when they find that the original result is flawed, they still may have trouble getting their paper published. The reason is surprisingly mundane: journal editors typically prefer to publish groundbreaking new research, not dutiful replications.
In March, for instance, Daryl Bem, a psychologist at Cornell University, shocked his colleagues by publishing a paper in a leading scientific journal, The Journal of Personality and Social Psychology, in which he presented the results of experiments showing, he claimed, that people’s minds could be influenced by events in the future, as if they were clairvoyant.
Three teams of scientists promptly tried to replicate his results. All three teams failed. All three teams wrote up their results and submitted them to The Journal of Personality and Social Psychology. And all three teams were rejected — but not because their results were flawed. As the journal’s editor, Eliot Smith, explained to The Psychologist, a British publication, the journal has a longstanding policy of not publishing replication studies. “This policy is not new and is not unique to this journal,” he said.
As a result, the original study stands.
Even when follow-up studies manage to see the light of day, they still don’t necessarily bring matters to a close. Sometimes the original authors will declare the follow-up studies to be flawed and refuse to retract their paper. Such a standoff is now taking place over a controversial claim that chronic fatigue syndrome is caused by a virus. In October 2009, the virologist Judy Mikovits and colleagues reported in Science that people with chronic fatigue syndrome had high levels of a virus called XMRV. They suggested that XMRV might be the cause of the disorder.
Several other teams have since tried — and failed — to find XMRV in people with chronic fatigue syndrome. As they’ve published their studies over the past year, skepticism has grown. The editors of Science asked the authors of the XMRV study to retract their paper. But the scientists refused; Ms. Mikovits declared that a retraction would be “premature.” The editors have since published an “editorial expression of concern.”
Once again, the result still stands.
But perhaps not forever. Ian Lipkin, a virologist at Columbia University who is renowned in scientific circles for discovering new viruses behind mysterious outbreaks, is also known for doing what he calls “de-discovery”: intensely scrutinizing controversial claims about diseases.
Last September, Mr. Lipkin laid out several tips for effective de-discovery in the journal Microbiology and Molecular Biology Reviews. He recommended engaging other scientists — including those who published the original findings — as well as any relevant advocacy groups (like those for people suffering from the disease in question). Together, everyone must agree on a rigorous series of steps for the experiment. Each laboratory then carries out the same test, and then all the results are gathered together.
At the request of the National Institutes of Health, Mr. Lipkin is running just such a project with Ms. Mikovits and other researchers to test the link between viruses and chronic fatigue, based on a large-scale study of 300 subjects. He expects results by the end of this year.
This sort of study, however, is the exception rather than the rule. If the scientific community put more value on replication — by setting aside time, money and journal space — science would do a better job of living up to Carl Sagan’s words.
Carl Zimmer writes frequently for The New York Times about science and is the author, most recently, of “A Planet of Viruses.”
WPI Says No to Retraction Request/ Levy Study Dashes Hopes /NCI Shuts the Door on XMRV
by Cort via Phoenix Rising Forums
Published on June 1st, 2011 06:01 AM
WPI Response - Dr. Mikovits response to the request for retraction started off with a blast -at none other than the editor in chief of the most influential science journal in the world over what appeared to be a relatively minor issue - a day or so breach in a papers embargo - which she called “gross disregard for the integrity of the scientific process”
- Dr. Mikovits, on the other hand, appeared to have good reasons to be upset. In a Science Now article she noted that the request for retraction came on a Thursday afternoon before a holiday weekend leaving her and the others “pretty well stunned," . The WPI knew that two more negative papers were coming out - virtually every body knew that - but with Science set to publish in 6 days, and without the new papers in hand, she clearly felt squeezed. It didn’t help that the news leaked a day early - causing the publication process to move up.
- The big issue, though, was the Journals request for the Lombardi et al authors to retract the paper and its decision to append a “Expression of Concern’ stating that the results of the Lombardi et al study were most likely due to contamination. The Editor in Chief of Science, Dr. Bruce Alberts, stated “the results are now seriously in doubt”.
- Dr. Mikovits stated that all the original authors, except for one, refused to retract the paper. In a Bloomberg news article http://www.bloomberg.com/news/2011-0...forumid=331851 Dr. Lipkin, the lead researcher in one of the NIH studies, stated that he felt that calls for a retraction were premature. Both NIH studies will continue with a due date by the end of the year.
- Chilling Effect - Requesting the retraction and adding the “Expression of Concern” to the study was a serious matter that will have a chilling effect on the field but how chilling isn’t clear…as the research community is well aware of the issues surrounding XMRV. Dr. Mikovits, though, asserted that the Expression of Concern would have ‘a disastrous effect on the future of this field”. Annette Whittemore, President of the WPI, stated “We are extremely disappointed that the editor of Science has published an ‘editorial expression of concern,’”
- Science could have simply retracted the article themselves but chose not to do so - putting themselves in something of logical impasse by asking the authors to retract the study but refusing to do that themselves. Instead the Science journal will wait, as others are, for the results of the all important NIH studies before they take any more steps.
- Major Response - It was not surprising, to see Dr. Mikovits produce her fullest response yet. In a long letter to Science she went over the original studies numerous accomplishments , suggested that many gammaretroviruses may very well have emanated from human tissue/mouse tissue culture experiments and then attempted to cast doubt on whether the studies suggesting contamination is present applied to the WPI.
- Dr. Mikovits pointed out the many ways she checked for contamination in the human cell lines, reagents and by using mtDNA. She accepted that contamination in other labs studies but can find no reason to assume that i occurred in her lab or the other labs in the study. Indeed, while negative evidence mounts, there is still no smoking gun…no direct path to contamination at the WPI.
- A Storm of Factors - There is no one issue that is turning members of the research against XMRV; instead it’s more of a series of factors that, put together, render XMRV in their minds a very unlikely candidate for disease. The new studies added more to the anti-XMRV argument but they were not definitive’. The new Paprotka paper was not new in the sense that that his data had already been reported in a Conference. The Levy study, not an exact replication study at all, added three things to mix; another well respected researcher came up negative using a variety of techniques, his reagent findings cast doubt on the Lo/Alter paper and his immune tests suggest that XMRV is quickly rendered inert by the immune system - making it difficult to see how it could cause disease.
- The Levy/Knox Study - With over 500 citations to his name, Dr. Levy may be the most prolific researcher yet to put his name on an XMRV study. A co-discoverer of HIV with a long history of gamma retroviral research he is well respected in the field. Lead author Dr. Konstance Knox has participated in several CFS pathogen studies. With researchers from Abbott Labs and the Blood Safety Research Institute (BSRI) also contributing the paper was top-heavy with experience.
- 56 participants in the study had tested positive for XMRV at the WPI. 19 of had blood drawn for the Levy study and the VIP Dx on the same day. Levy reported that he used the same methodologies reported in the Lombardi et al paper to look for antibodies and nucleic acids and as well as RT-PCR, culture (1-3 weeks) and chemoluminescence assays to look for antibodies. He also assayed XMRV’s ability to withstand attack from the immune system. Finally he assessed contamination in the reagents used in the Lo/Alter study and examined some of the original strains of XMRV.
- He was unable to find XMRV in any samples, found contamination in several of the reagents Lo/Alter used and in a twist showed that the complement part of the immune system was able to inactivate XMRV rendering it harmless suggesting that even if the virus was present in the human body that it would be able to do little. (Check out an excellent examination of this study at the CAA’s site here http://www.research1st.com/2011/06/0...-tribulations/
- The Propotka study showed how much hinges on XMRV’s genetic variability. That study concluded there was only a 1 in a trillion chance that the strain of XMRV found in the prostate cancer and WPI samples was not accidentally created in a lab some 20 years ago. The possibility that the WPI strains were inadvertently created in a lab is not a problem as labs can and have produced viral strains. The problem is that the strains from the WPI don’t bear any tell-tale remarks suggesting that they have been anywhere else than a lab. Viruses invariably change when they infect humans but the WPI (and prostate cancer) strains are almost exact duplicates of the original lab strains.
- WPI’s Submissions to GenBank Fall Short - Given that its been critical for the WPI to establish that their unpublished isolates display more variability. For over a year they have asserted that and used it as a basis for their claim that VP62 is not a suitable clone to use in research studies. (The last page of the supplemental science paper VP62 (S3) includes a photo showing that VP62 was used in one section of their original paper.)
- The CAA reported that Knox et al’s detailed examination of three sequences from the WPI lead them to conclude that they were similar to the laboratory strain (22RV1) under question. The paper, in fact, stated they were remarkably similar... “Therefore, the remarkable conservation of the WPI-XMRV sequences is most consistent with laboratory contamination with the original infectious VP62.” The CFIDS Association also reported, that a examination of the 18 sequences the WPI supplied to GenBank (which are freely available) indicates that they, too, are virtually identical to the past sequences.
- NCI Analysis of Original XMRV Samples Indicated They Were Likely Contaminants - On their website the NCI reported they have cultivated XMRV from the original samples and upon testing them concluded they too are so genetically similar to the lab strain that they, too, are probably laboratory contaminants.
- In a rather devastating but small study that does not appear to have been published yet the NCI also showed they could find XMRV in patient samples from the WPI and then went back and retested the same patients (without having their blood go through the WPI) and were unable to find any XMRV. This, of course, suggests that the patient samples picked up XMRV (were contaminated) as they passed through the WPI - the same conclusion the Shin/Singh study reached.
- The Science Journal’s reaction, then, reflected a series of factors. Dr. Coffin, once a avid supporter of XMRV stated “Taken together, these results close the door on XMRV as a cause of human disease.” These researchers clearly believe that the weight of the evidence obviates the need to find a ‘smoking gun’; ie discover the source of contamination at the WPI. (The WPI does not appear to have asked outside researchers to examine its operations).
- Complete Replication - The contamination argument is an important one but it is indirect. The main issue for XMRV may still be the inability of several dozen labs now, including some top labs in the US, to find XMRV in their blood samples. Many reasons for that have been asserted in the past but only one is left now-the lack of precise and complete replication - on the original study. In her response Dr. Mikovits stated:
- This would suggest that the methods and materials used in the non-replication studies are insufficient to use when attempting to detect human gammaretrovirus in the blood of human samples. The methods, processes, and materials of Lombardi et al. need to be followed precisely. The Alter and Lo study is the only study which has attempted a partial replication of the methods and materials of the Lombardi study, which confirmed evidence of MLV related viruses”
- Dr. Mikovits statement that the Alter and Lo study is the only study which has attempted even a ‘partial replication’ of the methods and materials of the Lombardi study will undoubtedly raise some hackles but Annette Whittemore released a chart indicating that this was so. The CAA’s analysis suggested that a complete replication study was impossible because researchers, as a matter of course, will never use materials from the original lab. They also noted that as techniques evolve, and they have evolved greatly since the original paper, researchers will use better techniques. (In fact they may not get published if they do not).
- Most of the studies have directly referred to the original Lombardi paper - that is, after all, the paper they are basing their studies on but it appears that researchers not only place different degrees of importance on the importance of ‘exact replication’ - but are using the term ‘replication’ in different ways.
- This was manifested most directly in the exchange regarding the Levy study with Dr. Mikovits stating “"They didn't do one thing we did,"and Dr. Levy stating, "We did it exactly the way they did it." One suspects they are both right in their minds…Dr. Mikovits found that Levy didn’t do any one experiment exactly as she did and Dr. Levy may believe that based on his 40 years in the field that he did everything he considered essential exactly as the WPI did.
- The WPI’s new document explaining exactly which tests have not been replicated is a long one. They also asserted that Lo/Alter used methods that were far closer to the WPI’s methods than anyone else. (The CAA’s Knox et. al review noted that some experts disputed the WPI’s assessment.) Here are some of the tests they mentioned.
The Conundrum Continues -The exact replication argument does not, however, seem to be swaying many researchers. Judging from the media reports a significant portion of the research community, including now the National Cancer Institute, the editors of Science, Dr. Levy, Dr. Knox and Dr. Raccaniello believe XMRV is either on life support or is dead. (The National Cancer Institute updated their website to state that “ The association of XMRV with cancers and diseases in humans has now been proven to be due to contamination from laboratory experiments”. ) These researchers have parsed the ‘exact replication’ argument and found it lacking in the face of several dozen negative CFS and other studies. If XMRV was there they feel that somebody at some point would have found it by now. One suspects that the exact replication scenario either has not happened in their experience or has happened very rarely. Dr. Stoye - who penned a positive assessment of XMRV early on perhaps expressed their mindset well; “Tens of labs have tried to reproduce their findings without success," Stoye says. "There are some very smart people in this, and they would not have got this wrong. It's an insult to us all.” A Uphill Battle - Dr. Mikovits started her career successfully overturning perceived opinion and she is trying do so again. She still has the support of Dr. Ruscetti and her co-authors and probably others. The June Retrovirology conference coming up shortly will undoubtedly be a heated one with NCI researcher and Lombardi et. al. co-author Dr. Ruscetti chairing one panel on XMRV shortly after his Institute publicly closed the door on it and the results of numerous XMRV studies on the docket. In an important presentation Lombardi et al. co-author, Dr. Silverman, will report on whether XMRV has contaminated samples or actually infects humans. Besides the Lipkin and BWG studies several studies are still outstanding including the Glaxo Smith Kline/CAA study, the Maldarelli study which is sending samples to the WPI and the Joliceur study. The Good News - The only good news is that as difficult as the situation is for the WPI and the XMRV finding, the Lipkin and BWG studies will go forward, giving them the opportunity to redeem XMRV and turn things around. In Amy Dockser Marcus’s latest blog, Dr. Ian Lipkin, provided a bit of calm in the storm stating "There are many instances where people are unable to replicate others' work. We need to see whether [the Whittemore Peterson institute] can replicate their own work with blinded samples. I think they should have the opportunity to do that.'' Silver Linings - One positive that has come out of this has been the almost unanimous endorsement from virtually every researcher that whatever happens with XMRV, ME/CFS needs more research. Chronic fatigue syndrome remains in the news like never before and has attracted the attention of the Director of the NIH, several Lasker Award winners and other luminaries. Funding levels for CFS outside of XMRV have not changed but the CAA reported that they believe that the extraordinary funding for XMRV has ‘laid the foundation for a national clinical-laboratory network that can provide the impetus for further research on CFS etiology, diagnosis and treatment.’ Whatever the fate of XMRV if it can do that it will have done well indeed.
- isolation of PBMC’s not PBL’s (only WPI)
- use of Ack Lysac buffer
- culture of ‘primary cells’ (only WPI did this)
- use of sodium Heparin blood tubes (only WPI)
- serology using Baf cells
- Western Blot of cultured primary cells (only WPI)
- Western blot of co-cultured cells (only WPI and Singh),
- Gag and env primers
- Nested PCR for gag
- Same Amplification methods
via Whittemore Peterson Institute; May 9, 2011
While WPI researchers continue to review the data presented by Dr. Singh, we believe that it is important to correct and clarify information regarding this study. Several individuals were consented to participate in this study as positive controls to enable Dr. Singh to develop assays to detect multiple variants of XMRV. Of these, only three were from the original Lombardi et al. cohort, two of whom were among those positive for a XMRV. A XMRV was isolated from one of those patient's PBMCs, cloned and fully sequenced (GenBank® accession number GQ 497343 as identified in the NIH genetic sequence database). Sequence data demonstrates that this virus is clearly distinct from XMRV (vp62) and 22Rv1. A budding virus particle from that sample was pictured in an electron micrograph in Lombardi et al. Virus from that patient sample was also transmitted both from the PBMCs and plasma to an uninfected indicator cell line, LNCaP. Finally, these results were supported by a separate lab using serological methods as reported by Lombardi et al.
Twelve additional samples from individuals not included in the Lombardi et al. study were independently collected by a third party and sent directly to Dr. Singh’s lab. Some of these subjects were positive for highly related sequences, including the polytropic and modified polytropic sequences identified by Lo et al., as determined by the WPI prior to the publication of the Singh study. Many of those subjects were also positive for ENV antibodies to a XMRV (vp62 and other XMRV family members), indicating that these patients had an immune response to a XMRV.
In addition, WPI investigators and others have provided evidence of sequence diversity between a XMRV (vp62), other similar XMRVs detected by WPI (designated internally with a number corresponding to a clinical isolate), a XMRV (p variant), and other related human gamma retroviruses. Therefore, we believe that it is vitally important that investigators interested in furthering the understanding of blood borne XMRV as a human pathogen use a proven positive clinical isolate as the control when developing
tests to detect this newly discovered human retrovirus.
WPI and the U.S. clinical laboratory performing XMRV tests pursuant to a license agreement with WPI have extensive controls in place to prevent and detect contamination. Approximately three thousand tests have been performed on patient samples to date using clinically validated tests; about one third have been found to be positive. Multiple sequences from these three thousand samples have been submitted to GenBank® and are awaiting publication. It is critical, in light of these findings, that all
treatment decisions are left to physicians and their patients, including the use of antiretrovirals.
While WPI researchers acknowledge that there is still much to be learned about the lifecycle and in vivoreservoirs of this family of human gamma retroviruses, we remain confident in the results reported in Science by Lombardi et al. Most importantly, we are committed to human gamma retroviral research in neuro-immune disease and will continue to offer our help to the medical and scientific community when requested.
by Cort via Phoenix Rising Forums
Published on May 5th, 2011 12:50 PM
- Dr. Singh’s study was one of the ‘biggies’ left. A murine retrovirologist at the University of Utah and the ARUP labs who had been studying (and finding) XMRV in prostate tissues, Dr. Singh started her CFS study about a year ago. Not only did it involve an expert in the field but Dr. Singh was working in tandem with one of our top ME/CFS physicians, Dr. Bateman and the Lights; it was an exciting study.
- The study was designed to account for many of the confounding factors such as patient characterization (CFS patients or not?), geographic locations (different variants of XMRV), clinical samples used (both healthy control and patient samples treated the same), blinding and other methods - which could have contributed to the negative results in other studies. The study was impressive on and an August 2010 article questioned whether it was, in fact, the best XMRV study underway? (See “The Best XMRV Study? Dr. Singh Talks!” .
- There were a number of encouraging early hints. First an initial smaller study was turned into a much larger second study - a seemingly clear signal that something had been found. A patent filed by Dr. Singh in association with ARUP indicated that indeed she had found XMRV and not just in CFS but in breast cancer and other disorders and in several different types of tissues and had gone so far as to distinguish antibody tests that did and didn’t work well. In an interview she stated that, given her extensive work with MLV’s, she wasn’t surprised at all that other researchers were having so much trouble. The stage seemed set for her to validate the WPI’s results.
- But then the study dragged on and on…and some hints suggested that the issues were more complex than she had envisioned. It was difficult to tell, given the tight-lipped approach taken by all the participants if there were big problems or if Dr. Singh just taking a really comprehensive approach to a complex subject? Both it turned out were true. Dr. Singh did believe she found XMRV early on but a closer examination suggested that contaminated reagents/robotic equipment were the cause and the study was large and complex (a 36 pager) with no less than 9 tests looking for XMRV.
- Amy Dockser Marcus reported Dr. Alan Light, one of the co-authors of the study, stated “We basically did everything we could think of to come up with a positive result”.
- Clearing Up Past Problems - In the introduction of the study Dr. Singh noted a plethora of problems in earlier studies -some of which had not, to my knowledge, been noted before. They included, in addition to those noted earlier small numbers of controls, lack of blinding, unsure limits of detection, lack of culture tests, lack of controls designed to flag contamination, low numbers of negative controls, lack of positive XMRV patients. This study eliminated all of those considerations and in his blog Dr. Racaniello called it the “most comprehensive study to date”.
- Size and Shape - With 105 people with CFS and 200 healthy controls and 14 people who had repeatedly tested positive for XMRV by the WPI this was a large study. The cohort, provided by Dr. Bateman, was a good one with many seriously ill patients who must have matched up well with those in the WPI study. Sixty-eight percent were females and all but five met both the Fukuda definition and the Canadian Consensus Criteria. They were not fat (?) (BMI 23.5) and the average age was 34.6. Most (72%) had an infectious onset and with an average duration of 16 years, most had been sick a long time. About 15% reported a family history of ME/CFS/Fibromyalgia.
- Functional assessment questionnaires indicated that they had high levels of physical disability that impaired them in every sphere of their life.
- Methods - Dr. Singh developed her own qPCR and antibody tests and used several of the tests used in the original Lombardi paper and the Lo/Alter paper to test the Utah patients. (In an interview Dr. Singh explained qPCR tests cut down the possibility of contamination because, unlike the nested PCR, it was only run once.) She used her qPCR tests to hone in on four different regions of the virus and adapted, with Dr. Frank Ruscetti’s ‘extensive help’ the WPI’s original culture test (Dr. Ruscetti was a co-author of the original paper). The culture test lasted, as per Dr. Mikovits instructions, six weeks.
- Dr. Singh used the nested PCR protocol (gag sequence) in the original Lombardi paper and the protocol Alter/Lo used to test for XMRV/MLV’s in the 14 patients who had repeatedly tested positive at the WPI as well as all her tests. The positive patients, then, were tested for XMRV in nine ways; 4 qPCR tests developed by Dr. Singh, two antibody tests, a Ruscetti culture test, nested PCR (gag) (Lombardi) and nested PCR (Lo/Alter).
- Results - She was unable to detect XMRV or MLV sequences (aka the Lo/Alter study) in any of the Utah ME/CFS patients, the positive patients provided from the WPI or the healthy controls samples using any of these tests.
- Dr. Racaniello strongly endorsed Dr. Singhs results stating, "Given the care with which these numerous assays were developed and conducted, it is possible to conclude with great certainty that the patient samples examined in this study do not contain XMRV DNA or antibodies to the virus."
- WPI Test Produces Some False Positives - When Dr. Singh used the PCR assay used in the Lombardi paper and the PCR assay used in the Alter/Lo paper, however, about 5% of healthy controls and people with ME/CFS tested positive. Questioning whether the test might have picked up a laboratory produced plasmid she did a qPCR test that could determine whether she had found a wild type virus or the infectious clone she had produced to run the tests. The test indicated she had not picked up her laboratory plasmid.
- She then created a ‘remarkably sensitive’ qPCR test that looked for IAP sequences to check for mouse DNA and didn’t find any mouse DNA either!
- Had the WPI’s assay really picked up XMRV (albeit in low levels in both CFS patients and controls)? After noticing an odd pattern; upon retesting the 5% level of positive results remained the same but that different samples were testing positive she dug deeper.
- Contaminated Reagents - First Dr. Singh tested 36 uninfected LnCaP cells used for culturing and found two of them were positive for XMRV using the WPI’s nested PCR but not with her qPCR or the IAP test. (Since they had not been infected with XMRV all should have been negative). Then she began to test different components of the actual test itself and found that two enzymes (Taq polymerase and Platinum Taq polymerase from Invitrogen) tested positive for mouse DNA. When she began testing different batches of the polymerases she found that four different batches were contaminated. Repeated testing of another polymerase from Amplitaq, on the other hand, indicated that it was clean.
- A Different Course of Contamination - The authors noted that the ‘detection of XMRV in the blood is fraught with difficulties” and added their own strange story of accidental contamination to the mix. They initially used a biorobot that had, some months earlier, been used to extract DNA from XMRV positive tissue cultures. Despite using ‘sterile, disposable reagents’ traces of XMRV, to their surprise, still lingered in the biorobot several months later and ended up producing the early false positives. Once Dr. Singh abandoned the biorobot (in this case men were better than machines) the positive results stopped. On a positive note, for their study, they noted that their ability to find even the trace amounts of XMRV left behind indicated how sensitive their tests were.
- The authors believe contamination was probably responsible for the Lombardi papers results. They noted that culturing, the method that has worked best at the WPI , was ‘especially vulnerable to contamination’. Their ability to find mouse DNA in a PCR reagent which was developed using a mouse monoclonal antibody was not surprising but mouse DNA’s appearance in a reagent developed without mice was surprising.
- They followed the lead of some other researchers in proposing IAP tests were the best contamination test. Except for handling (ie testing) samples more frequently they were unable, however, to come up with a good explanation for the strikingly different PCR results between the healthy controls and the patients in the original study. They noted that all ‘possible - and seemingly not possible’ sources of contamination (reagents, equipment, etc.) should be checked.
- They also noted, however, that serological (ie antibody) results are not nearly as susceptible to contamination but are susceptible to false positives if they cross-react to other pathogens or other proteins. Their two antibody tests did not reveal XMRV was present and they suggested this meant the positive results in the original study may have been due to a cross-reaction to another virus or protein and not to XMRV.
- Strong Conclusions - Drawing on the negative tests of the formerly positive patients the authors went so far as to say that they ‘feel that XMRV is not associated with CFS’ not just in Utah but anywhere, and therefore felt ‘forced to conclude’ that prescribing antiretroviral drugs is ‘insufficiently justified’ and ‘potentially dangerous’.
- They also noted that there is a ‘wealth of data’ suggesting infectious involvement in ME/CFS and, that efforts in that area should continue.
Where is XMRV Now?
- Given the lack of evidence for XMRV or XMRV-like viruses in our cohort of CFS patients, as well as the lack of these viruses in a set of patients previously tested positive, we feel that that XMRV is not associated with CFS. We are forced to conclude that prescribing antiretroviral agents to CFS patients is insufficiently justified and potentially dangerous.
- The most problematic finding for XMRV is not that researchers are finding it but declaring it to be a contaminant (although that has happened in three published studies and one unpublished one) but that once again, they are simply unable to find evidence of the virus even in low levels in CFS patients and other disorders. At this point the question where it came from - the product of a laboratory accident or an escape from mice - is somewhat irrelevant. Researchers have to find the virus in people with ME/CFS before they can associate it with the disease.
- Since the Lombardi paper was published in Oct. 2009 none of the 13 ME/CFS studies and only 1/14 studies on other disorders (not including prostate cancer studies) have found XMRV. The really hard fact to confront is that none of the 24 studies that have looked XMRV in the blood in over the past year and a half in 9 different disorders since the Lombardi paper have found any XMRV at all.
- Unpublished reports of positive studies include the WPI's London study, Dr. Meirleir’s study and Dr. Cheney’s VIP Dx study. Unless they are done by reputable antiretroviral labs, however, the De Meirleir and Cheney findings, at this point would probably count for very little when specialists in the field are unable to find the virus.
- The Big Question remains why the WPI was able to find XMRV in such high numbers in the Lombardi study of Oct 2009 and why few others have. The lists of proposed reasons XMRV was not being found is a long one and includes the wrong kinds of patients, wrong tests, not using culture, incorrect storage procedures, incorrect sampling procedures, freezing/refreezing samples, no antibody tests, wrong annealing temperatures, not calibrating magnesium levels….All of these have been more or less definitively at one time or another stated to be the reason or a major reason why XMRV was not being found.
- Almost all of those have been resolved. The sampling/storage questions were resolved in the second part of the BWG study. The Satterfield study addressed questions with PCR (annealing temperatures, magnesium levels) and antibodies. The Singh study was able to cross off wrong patient cohort, wrong tests, not using culture, geographical differences in viral spread or genetic variation. Culturing was a big issue but Dr. Singhs culture tests created in ‘extensive’ collaboration with Dr. Ruscetti suggested that lack of culturing was not the answer either.
- What is Left? A good question is ask now is what other than contamination that could possibly explain the differing results? Not using a wild-type virus to calibrate the PCR test has been put forward but the CAA article titled the “The Iteration of X” which noted that Dr. Singh’s clone, which was a combination of two VP62 plasmids, actually produces an infectious, wild-type virus in culture would seem to eliminate that argument.
- A Smaller Bench - The strength of the original study has long since faded with researchers finding ways to explain each of the findings away. (The disparate results between the healthy controls and patients is still the most difficult to explain). Dr. Coffin, an early supporter is gone. It was hoped that Dr. Singh, a specialist in these viruses, would save the day but that hasn’t happened. Dr. Racaniello has been careful to leave the door open but went so far this time as to imply that the research community has made up its mind on XMRV and that the WPI should track down the source of contamination in their lab so that the issue does not linger in the ME/CFS community.
- The CFIDs Association article suggested that the research community is not so convinced as that as they reported that the two big studies underway, the Lipkin and BWG study, will continue. The results of the BWG study should be in by the end of summer and the Lipkin study, which is still gathering approvals, will take much, much longer.
- XMRV’s bench, so to speak, is getting smaller and smaller yet it does still contain important players in retroviral research. None of the co-authors of the original study which included Dr. Silverman, Dr. Ruscetti and of course, Dr. Mikovits, have disavowed the original study results and both Dr. Mikovits and Annette Whittemore appear recently to be entirely confident about their results. It’s almost inconceivable that the WPI has not, in the face of all this controversy, been doing extra internal testing involving controls to ensure that they are on the right track. Those results could underlie their confidence.
- What could these studies still be doing wrong after all this time? That answer will have to come from the WPI and the original study authors. The WPI has not, as yet, made an official response to the Sin/Singh study.
- More to Come - Meanwhile there are still major studies to come. The Glaxo-Smith Kline/CAA study and University of Alberta studies are long overdue. The results of a Levy culture study are expected soon. Dr. Joliceur in Canada and the NCI are looking at WPI positive patients, and, of course, there are the two ‘biggies’; the BWG and Lipkin studies. ndly, if a patients tested positive, was this retested to confirm it or did they stop at that point? If, as Singh found, there were a low level of false positives, if you retested the negatives you'd get more positives, retest those negatives remaining to get more positives and so on unitl you ended up with a high level of positives for patients. But maybe WPI retested all their patient positives to confirm the results.
Ian Lipkin on XMRV
6 MAY 2011
Late last year virologist Ian Lipkin was asked by National Institute of Allergy and Infectious Diseases head Anthony Fauci to coordinate a multi-center study of CFS patients. Newly drawn blood samples from 100 CFS patients and 100 healthy controls from around the US will be blinded and sent to three groups – FDA, CDC and the Whittemore Peterson Institute – and assayed for the presence of XMRV. After the recent publication by Ila Singh on XMRV in CFS patients, Dr. Lipkin sent me the following note:
We have a plethora of explanations for how CFS/XMRV/MLV studies could go awry. However, we don’t have evidence that they have. Absent an appropriately powered study representing blinded analyses by Mikovitz and Lo/Alter of samples from well characterized subjects using their reagents, protocols and people, all we have is more confusion.
I remain agnostic. We won’t have answers until the end of 2011.
The NIH will post something on our study today.
Another XMRV/MLV study bites the dust ... every study that WPI is involved in seems t
Clifford H. Shin1, Lucinda Bateman2, Robert Schlaberg1, Ashley M. Bunker3, Christopher J. Leonard1, Ronald W. Hughen4, Alan R. Light4, Kathleen C. Light4, and Ila R. Singh1,*
1 Department of Pathology, University of Utah, Salt lake City, Utah, 84112
2 Fatigue Consultation Clinic, Salt Lake City, Utah, 84102
3 ARUP Laboratories, Salt Lake City, Utah, 84108
4 Department of Anesthesiology, University of Utah, Salt Lake City, Utah, 84112
* Corresponding author: Mailing address: Emma Eccles Jones Medical Research Building, Department of Pathology, 15 North Medical Drive East, Suite #2100, Salt Lake City, UT 84112, Phone: (801) 213-3737, Fax: (801) 585-7376, Email: email@example.com
Chronic fatigue syndrome (CFS) is a multi-system disorder characterizedby prolonged and severe fatigue that is not relieved by rest.Attempts to treat CFS have been largely ineffective primarilybecause the etiology of the disorder is unknown. Recently CFShas been associated with xenotropic murine leukemia virus-relatedvirus (XMRV) as well as other murine leukemia virus (MLV)-relatedviruses, though not all studies have found these associations.We collected blood samples from 100 CFS patients and 200 self-reportedhealthy volunteers from the same geographical area. We analyzedthese in a blinded manner using molecular, serological and viralreplication assays. We also analyzed samples from patients inthe original study that reported XMRV in CFS. We did not findXMRV or related MLVs, either as viral sequences or infectiousvirus, nor did we find antibodies to these viruses in any ofthe patient samples, including those from the original study.We show that at least some of the discrepancy with previousstudies is due to the presence of trace amounts of mouse DNAin the Taq polymerase enzymes used in these previous studies.Our findings do not support an association between CFS and MLV-relatedviruses including XMRV and off-label use of antiretrovirals for the treatment of CFS does not seem justified at present.