Science 23 September 2011:
Vol. 333 no. 6050 pp. 1694-1701
Virology False Positive
A report in Science 2 years ago that linked a mouse retrovirus, XMRV, to chronic fatigue syndrome astonished scientists and patients alike. But the theory soon began to take hits, and now, to all but a few researchers, it has completely unraveled.
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CREDIT: M. HICKS/SCIENCE Done. Case closed. Finito, lights off, The End.
For the past 2 years, a controversy has roiled around the purported link between a mouse retrovirus, XMRV, and chronic fatigue syndrome (CFS), a baffling, debilitating disease with no known origin. Many researchers who have followed this saga closely thought that a definitive study, published online this week by Science (http://scim.ag/xmrv-cfs
) and conducted by nine labs—including the main proponents of the thesis—would finally bring a halt to the impassioned debate.
The uproar began with an October 2009 paper in Science that found XMRV in the blood of two-thirds of the CFS patients examined. A steady assault on the report soon began, with more than a dozen labs failing to replicate it to date and several asserting that contamination must have occurred. The leader of the team that conducted the study, Judy Mikovits of the Whittemore Peterson Institute for Neuro-Immune Disease (WPI) in Reno, Nevada, resolutely maintained that her lab had no evidence of contamination and that it could repeatedly find the virus with its techniques. Millions of dollars have gone into clarifying the question, which has had far-reaching consequences for people with CFS and, if the virus lurked in the blood supply, the public at large.
The study just published found that none of the nine labs could reproducibly detect XMRV or relatives of the virus in blood samples distributed under a blinded code. Pounding another nail into the coffin, Science is also running a partial retraction (http://scim.ag/R-H-S
) of the original paper, as a contributing lab found that it in fact had a contamination.
In an unexpected twist to this operatic saga, Mikovits co-authored the Science Express paper and has no quarrel with the results. Her collaborator, Francis Ruscetti, a retrovirologist at the U.S. National Cancer Institute (NCI) in Frederick, Maryland, who is a co-author of both the original Science report and the new one, concurs. “Where there is disagreement is in the interpretation,” Ruscetti says.
By their lights, the new study—conducted by the Blood XMRV Scientific Research Working Group sponsored by the U.S. National Heart, Lung and Blood Institute—does not rule out the possibility that mouse retroviruses infect people with CFS. “The conclusion of the Blood Working Group was that we don't have a reproducible assay to detect XMRVs in the blood—not that they weren't in the patients at all,” Mikovits says. Ruscetti adds that the working group analyzed their original patients but used samples taken a few years later.
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CREDIT: J. COHEN/SCIENCE If this seems like wordsmithing and splitting hairs, welcome to the confusing, maddening world of XMRV. Mikovits and Ruscetti, who have become increasingly isolated from the broader scientific community, now say their original paper erred by focusing on a single XMRV isolate that turned out to be a contaminant. They say that isolate is but one of many XMRVs, which belong to a still larger family of gammaretroviruses. They also contend that the virus may lurk in tissues, only traveling to the blood occasionally. “We still stand by our data that we isolated gammaretroviruses from patients with CFS and also from healthy controls,” says Mikovits, who has taken a more public role than Ruscetti in battling critics and reaching out to supporters.
Mikovits has become something of a savior in the community of people with CFS (also known as myalgic encephalomyelitis, or ME), who for decades have endured charges that the disease is psychosomatic. The 2009 Science paper shouted out that CFS may well have a clear biological cause, and, in turn, raised hopes of effective treatments and even a cure. The new findings give her “great pause,” yet she suspects they're but a speed bump. “I haven't changed my thinking at all,” she says. And she worries that the Blood Working Group conclusions will confuse people with CFS, some of whom got wind of the results early in the blogosphere and contacted her in a panic. “I had 15 suicidal patients call me last week,” she says.
In scientific circles, Mikovits has developed a less flattering reputation. Critics have accused her and her backers of stubbornly wedding themselves to a thesis and moving the goalposts with each study that challenges their conclusions. Even disease advocates who welcome the attention XMRV has brought to CFS believe the time has come to put this line of research to rest. “It's hard to say that this has not received a fair appraisal,” says Kimberly McCleary, president of the CFIDS Association of America, a patient group in Charlotte, North Carolina.
Many retrovirologists wish the entire controversy, which has ensnarled dozens of labs and cost millions of dollars, would simply disappear. “All of it's a waste of money and it's wrong,” says Robert Gallo, head of the Institute of Human Virology in Baltimore, Maryland. “It's like a bad dream.”
XMRV owes its discovery to a little-known enzyme called RNase L that helps the body battle viruses—and has ties to both prostate cancer and CFS.
In the early 1990s, a few CFS researchers reported that their patients had higher levels of RNase L than healthy people had, suggesting that this natural viricide signaled an undiagnosed infection causing the disease. RNase L levels, they contended, provided a long-sought “biomarker” for CFS, which has caused much confusion because clinicians use varying criteria to make a diagnosis. It also could potentially help researchers gauge the effectiveness of treatments, as a drop in the enzyme would mark a decrease in underlying, undetected infections. This idea became central to tests using Ampligen, an immune modulator made by Hemispherx Biopharma in Philadelphia, Pennsylvania, to treat CFS.
One of the clinicians testing Ampligen was Daniel Peterson of Incline Village, a town on the northern shore of Lake Tahoe, the popular tourist destination on Nevada's border with California. Peterson had investigated a mysterious outbreak of CFS that began in Incline Village in 1984 and had become one of the world's best-known CFS clinicians. His work drew the attention of Harvey Whittemore, a prominent Reno attorney who lobbied on behalf of the gaming industry and later became a major real estate developer, and his wife, Annette, a teacher of children with cognitive disorders. The Whittemores' daughter, Andrea, had developed CFS at age 12, and at Peterson's suggestion she began receiving Ampligen.
With the Whittemores' financial support, Kenny De Meirleir, a Belgian physician who had an RNase testing lab in his own country, helped them open RedLabs USA in Reno to monitor the enzyme in U.S. patients taking Ampligen. Vincent Lombardi, who would soon play a prominent role in the XMRV saga, co-founded the new lab and served as its director of operations.
Lombardi was a late bloomer in science. In 1990, while he worked as a securities trader in Lake Tahoe, he decided to pursue an undergraduate degree at a local school. As part of a biostatistics class, he worked with Peterson, analyzing immune parameters of CFS patients. After his graduation in 1995, he ran investment companies and also worked as a marketing director for a pawnbroker business. He went on to pursue a Ph.D., first studying RNase L but switching to peptides in the tobacco hornworm, which he completed at the University of Nevada, Reno, 2 years after starting RedLabs USA.
In 2002, meanwhile, a team of prostate cancer researchers had discovered that a mutation in the RNase L gene frequently occurs in families in which men are prone to the early-onset form of that disease. A few years later, one of those investigators, Robert Silverman of the Cleveland Clinic in Ohio, began probing for a viral link: If RNase L fights viruses, he reasoned, then perhaps having a crippled form of the enzyme opened the door for a cancer-causing infection.
Silverman teamed up with Joseph DeRisi and Don Ganem, two veteran virus hunters at the University of California, San Francisco (UCSF), who had developed a microarray called ViroChip that had proved its mettle in identifying new viruses. The assay confirmed Silverman's hunch: In the prostate cancer tumors, ViroChip found a novel retrovirus, apparently a cousin of a known mouse virus and therefore given the ungainly name of xenotropic murine leukemia virus–related virus (XMRV).
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CREDIT: KELVIN MA/TUFTS UNIVERSITY Silverman first reported the existence of XMRV and its prostate cancer link in April 2005 at an HIV meeting in a mountain resort in Banff, Canada. “My talk was well received, people were clearly interested, but it did not receive that much attention,” Silverman wrote to Science in an e-mail delivered through a public affairs manager—the only form of communication the Cleveland Clinic would allow. (XMRV has become such a radioactive topic that several institutions restricted what their researchers could discuss, and both Ganem and DeRisi declined to talk to Science about their XMRV work.)
In March 2006, Silverman, DeRisi, Ganem, and colleagues published their report in PLoS Pathogens, a respected but decidedly low-key choice for the description of a new retrovirus that appeared to infect humans—the only three others are HIV and HTLV-I and -II—and had ties to a serious disease. Although they wrote that there was a “strong association” between XMRV and the RNase L mutation, the researchers still had misgivings about whether the virus contributed to prostate cancer.
In a commentary in the 30 January 2007 issue of the Proceedings of the National Academy of Sciences (PNAS), mouse retrovirologist Hung Fan of UC Irvine called it an “exciting” discovery. But others were skeptical. Gallo, whose lab played a central role in the discovery of all three known human retroviruses, contacted PNAS's editor to complain. The commentary was “substantially over the top,” says Gallo, who saw no compelling evidence that this supposedly new human retrovirus caused disease and doubted that a mouse retrovirus could even infect humans. “Once claims of etiology were made, I just gasped for breath,” Gallo says. “My own experience argued to me that it's best to stay away from this one.”
Around the time Silverman first publicly described XMRV, Annette Whittemore took on one of the most ambitious pursuits of her career: using her family's wealth and powerful contacts to build a full-fledged research and clinical institute devoted to CFS. Peterson would head a medical research team to elucidate the causes of the disease and provide state-of-the-art care. Nevada Governor Kenny Guinn and the current U.S. Senate Majority Leader, Harry Reid (D–NV)—both family friends of the Whittemores—helped arrange government funding, while the Whittemores' alma mater, the University of Nevada, Reno, offered about 1400 square meters of lab space within a new, gleaming $78 million Center for Molecular Medicine. It would take 4 years for that building to open, so the nascent Whittemore Peterson Institute (WPI) set up shop in borrowed space on campus in 2006. Annette, WPI's president, hired Mikovits to run the lab.
Mikovits had come into the CFS world by a circuitous route. She had spent more than 20 years at NCI, first as a technician studying HTLV-I and HIV with Ruscetti, who served as her Ph.D. adviser. In 2001, she married and moved to southern California, becoming chief scientific officer at EpiGenX Biosciences in Santa Barbara, which aimed to use a new epigenetic approach to develop drugs and diagnostics.
In 2006, Mikovits became a consultant to a CFS-related nonprofit foundation that Annette Whittemore co-founded, which explored the link between the disease and another virus, human herpesvirus 6 (HHV-6), that had been discovered in Gallo's lab. At a meeting in Barcelona, Spain, that year, Mikovits spoke to Whittemore for the first time and heard Peterson give a talk. Peterson focused on a non-Hodgkin's lymphoma in some of his CFS patients, and Mikovits smelled a virus. She offered to work with him, and Whittemore helped set up a collaboration. Later that year, Mikovits joined WPI.
She soon enlisted Ruscetti, who had worked in Gallo's lab when it discovered HTLV-I, to screen blood samples from Peterson's patients for viruses. Intrigued by the RNase L link to XMRV, Mikovits and Lombardi—who by then had joined WPI as well—met Silverman in October 2007 at a prostate cancer conference in Lake Tahoe, where they discussed the possible role of XMRV in CFS. Silverman was happy to collaborate and sent WPI a clone of the virus, known as VP62. The institute could use it as a reference to start hunting for the virus in CFS patient blood samples that Peterson had stored.
A little over a year later, on 18 November 2008, Mikovits had the first evidence of XMRV in CFS patients. Working with Ruscetti and Silverman, the group amassed evidence that the virus occurred in 67% of 101 CFS patients and 3.7% of 218 healthy controls. If the latter number was representative of the general population, up to 10 million healthy people in the United States alone might be infected, and the virus might be spreading through blood donations and organ transplantation—a silent epidemic of frightening proportions. Mikovits, Ruscetti, Silverman, and their co-authors submitted a paper to Science in May 2009. The paper, of which Lombardi was first author, did not claim that XMRV caused CFS, noting that the disease might simply make patients more vulnerable to infection. Causality “is probable but not definitive at this time,” Lombardi et al. stated.
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“Here was this mysterious disease, … and along comes a new virus. … It seemed to … have the makings of a medical breakthrough.”
—ROBERT SILVERMAN, CLEVELAND CLINIC
CREDIT: LISA DEJONG/LANDOV But what they were asserting was stunning enough: WPI, NCI, and the Cleveland Clinic had all found evidence of XMRV in the same CFS patients. Some of these people had participated in a 2007 NCI drug study, too, and their blood samples, stored separately, also tested positive, “ruling out the possibility of lab contamination as a source,” the authors wrote. When the team calculated the so-called p-value—which generally needs to be under 0.05 for a finding to be considered significant—they arrived at the astonishingly low number of 8.1 × 10−35.
Oddly, Peterson, who had supplied the patient samples, was not one of the authors. His name was left off—and he was kept out of the loop on the study's results—because of worries that he might prematurely tell his patients, Mikovits says.
The manuscript didn't convince Science. After reviews by three referees and members of the Board of Reviewing Editors (provided for this article by Mikovits), the editors rejected the paper. “Although the referees were intrigued by your findings, they had a number of serious reservations,” read a 4 June letter, which included excerpts from reviewers.
The rejection letter noted that Science would re-review the paper if the authors could both retain the “novelty of its main message” and “address the referees' concerns with new data rather than with counter-arguments.” But the criticisms were substantial. “Chronic Fatigue Syndrome is full of false alarms,” wrote one advisory board member, “and the detection of XMRV could be false positive PCR.” An otherwise enthusiastic referee wrote that the “one major caveat I have is that the issue of potential contamination has not been completely dealt with.” A second referee found it odd that the genetic sequence of XMRV derived from CFS patients and the virus earlier discovered in prostate cancer were 99% similar. This “seems very unlikely and may indicate contamination, despite the evidence presented to the contrary,” the referee warned. One also wondered why they omitted Peterson as a co-author.
Mikovits and her co-workers addressed many of the critiques, and on 14 July, they resubmitted the paper, this time with Peterson's name on it. The next week, a committee—which included John Coffin, a prominent retrovirologist at Tufts Sackler School of Graduate Biomedical Sciences in Boston, and Silverman—organized a workshop at NCI with Mikovits and Ruscetti to help the institute better understand the unanswered scientific questions and the potential public health ramifications. Dusty Miller, a retrovirologist at the University of Washington, Seattle, who had studied XMRV, left the meeting convinced that the link to CFS was real. The group had not only detected the virus using PCR but also grown it from patients' cells and found antibodies to it. “It sounded really good to me because they had all these different lines of evidence,” Miller says. The fact that an NCI veteran such as Ruscetti endorsed it helped convince him. “Frank said, ‘I grew it with my own hands,’” Miller recalls. “At the time, it sounded really exciting.”
Coffin, who chaired the meeting, thought Mikovits and her colleagues had persuasive evidence, especially because XMRV formed its own branch on a genetic family tree of mouse retroviruses, strengthening the case that it was not a contaminant.
The next day, as Mikovits sat at Washington, D.C.'s Dulles International Airport waiting for her flight back home, she received a phone call from Science, indicating for the first time serious interest in publishing the paper. Science accepted the Lombardi paper on 31 August and published it online on 8 October 2009, accompanied by a supportive commentary that Coffin co-authored. “There are several lines of evidence that transmission happened in the outside world and was not a laboratory contaminant,” the commentary declared. The publication catapulted Mikovits into the spotlight, where she has remained ever since.
“Here we go again.” That's what crossed Kimberly McCleary's mind when she read the first headlines about the paper that week in her office in Charlotte. In her 20-plus years at CFIDS, McCleary had seen many infectious agents fingered as a potential cause of CFS. HHV-6. Epstein-Barr virus. Mycoplasma. Adenovirus. Cytomegalovirus. HTLV-I and -II. And on and on. Each time, hopes were dashed as scientists closely evaluated the suspects.
The string of disappointments had made McCleary cautious, and CFIDS urged patients not to get carried away. “We tried to temper things early on, and we were criticized heavily for raining on the parade,” she says. Still, many patients went wild with joy. “ME/CFS patients have never seen anything like this,” wrote Cort Johnson in an item, “Game Changer,” posted on his popular blog Phoenix Rising.
Lombardi et al. had made XMRV a superstar. “Here was this mysterious disease (CFS-ME) with no known cause, all of these patients suffering, and along comes a new virus that associates with the disease,” Silverman says. “It seemed to some at the time to have the makings of a medical breakthrough.”
But many scientists were skeptical. Simon Wessely, a psychiatric researcher at King's College London who has long studied CFS, says the virology went over his head, but the fact that fully two-thirds of CFS patients harbored the virus was an alarm bell. CFS, whose definition has been the subject of years of debate, is far too heterogeneous a phenomenon for that, he says.
With a few U.K. colleagues, Wessely started penning a letter to Science to address what they saw as methodological flaws in the paper. One problem was the unclear patient selection, they wrote. If all patients came from Nevada and healthy controls were from elsewhere, then perhaps XMRV wasn't a CFS-related virus, Wessely says, but something that happened to be more prevalent in the gambling state.
Wessely also contacted Jonathan Weber, a retrovirologist at Imperial College London, to set up a study of British CFS patients. “Our reaction was, ‘It's probably wrong, but if it's true, it's a pretty big advance. So it's worth testing,’” he says.
The first published critique appeared a few weeks later—and it would prove to be prophetic. On 18 November, Patrick Moore, a cancer virologist at the University of Pittsburgh in Pennsylvania, and his postdoc Masahiro Shuda published a blistering commentary on Faculty of 1000, a Web site that evaluates reports in what it calls postpublication peer review.
“Unfortunately, in my field, there's a tendency with any new virus to hope that it is causing a disease,” says Moore, who co-discovered HHV-8 and helped prove that it causes Kaposi's sarcoma. “One has to be so cautious about that. A lot of stories sound good, but they're built on a house of cards.”
As Moore and Shuda noted, Mikovits's group used a technique called “nested PCR” to identify XMRV infections, which, they wrote, “is inherently prone to intermittent false positivity that has occurred in our lab and many others.” What's more, the researchers had not randomized and blinded patient and control samples, a standard way to protect against bias and to detect errors. Together, Moore and Shuda wrote, this was “a recipe for uncontrolled PCR contamination.”
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“We tried to temper things early on, and we were criticized heavily for raining on the parade.”
—KIMBERLY MCCLEARY, THE CFIDS ASSOCIATION OF AMERICA
CREDIT: COURTESY OF KIMBERLY MCCLEARY The next month, a study co-authored by Silverman and published online by Virology showed that AZT, an anti-HIV drug, worked against XMRV in test tubes. Again, CFS patients were overjoyed. Not only had a likely culprit of their suffering been found, drugs already on the market might treat it.
Of course, no one had proved that the virus actually caused CFS. But by the start of 2010, three commercial labs, one of which was WPI's VIP Dx (formerly RedLabs USA), offered an XMRV test.
Peterson, sitting in one of his practice's waiting rooms in Incline Village in June, explained that his patients split into two groups. One had ridden the CFS roller coaster so many times that these patients wanted validation and replication before buying the XMRV story. “There's another group that, for whatever reason, has made this illness into a religion and becomes polarized into who believes and who doesn't believe,” he said. Increasing numbers of believers began taking XMRV tests, which cost $500 or more, and, if they were positive, asking their physicians to prescribe anti-HIV drugs, says Peterson, who by then had severed ties with WPI over a contractual issue.
When a Science reporter visited WPI the same month, Annette Whittemore and Lombardi both strongly defended the decision to sell an XMRV test before evidence existed of a causal link. (The group has filed related patent applications, too.) “Every physician who requests patient testing is aware of contamination and the potential of false hope,” Lombardi said. “We're not the doctors ordering these tests for our patients.” Whittemore stressed that all profits would go back into the institute. “This wasn't set up to make money, and it never has,” she said. Whittemore also dismissed critics—a group that included Peterson— who cringed at CFS patients taking antiretrovirals. “How many years does this patient population have to be impacted and their lives destroyed?” she asked.
Andrea Whittemore was one of the CFS patients who tested positive for XMRV.
The flood of negative data started in January 2010, when Wessely, Weber, and their colleagues reported in PLoS ONE that they couldn't find any trace of XMRV in 186 British CFS patients. By the end of February, two more negative reports were published, one of them by Jonathan Stoye, Coffin's co-author on the favorable Science commentary that accompanied the Lombardi paper.
Back in Reno, Mikovits and Lombardi began feeling besieged. “After the first negative study, it was a dog pile,” Lombardi says. “Let's be honest: A number of people in the mainstream medical community heard chronic fatigue, and they rolled their eyes and laughed.”
The problem, as they saw it, was that nobody was following their recipe exactly. Many labs failed to find XMRV using PCR because the virus exists in scant amounts, they said—so much so that a patient can test positive on one bleed and negative on the next. Mikovits said they overcame this problem by first mixing patient blood samples with an uninfected cell line that is especially permissive to the virus and coculturing for 8 weeks. Other researchers, including XMRV discoverer Silverman, questioned the need for this step.
Seven months after Lombardi et al., Science published Wessely's critique and two others, which discussed patient selection problems and the growing list of negative studies, including three that failed to find XMRV in prostate cancer. None of the Technical Comments, however, mentioned the possibility of contamination.
Despite a vigorous defense by Mikovits and Ruscetti in the same issue of Science, the three comments further eroded confidence in the findings. But then, 2 weeks later, a research group unconnected to WPI would rekindle the fading hope that the link between XMRV and CFS was real. It would also spark a peculiar new controversy.
That study originated with Shyh-Ching Lo, an infectious-disease researcher at the U.S. Food and Drug Administration (FDA) who took a keen interest in CFS. In the early 1990s, Lo had tested a theory that a microbe called Mycoplasma was involved in CFS. For that study, he had received blood samples from Anthony Komaroff, a physician who had treated hundreds of CFS patients at Brigham and Women's Hospital in Boston. Like so many others, the lead had proved spurious.
After XMRV surfaced as a new candidate, Lo suggested to Komaroff they test the same patient samples, stored at −80°C and untouched for almost 2 decades. Komaroff was enthusiastic. Lo also contacted Harvey Alter, an infectious-disease specialist who works on transfusion medicine at the U.S. National Institutes of Health's Clinical Center in Bethesda, Maryland. Alter—who won the prestigious Lasker Award for his role in the discovery of hepatitis C—provided Lo with blood samples from healthy people to serve as controls.
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“Once claims of etiology were made, I just gasped for breath. My own experience argued to me that it's best to stay away from this one.”
—ROBERT GALLO, INSTITUTE OF HUMAN VIROLOGY
CREDIT: GAIL BURTON/AP IMAGES It was Alter who accidentally dropped the bombshell news. At a blood bank meeting in the Croatian capital Zagreb in late May 2010, he presented an overview of current blood safety issues, and his Power-Point presentation had one slide on what he called the “Agent du jour,” XMRV. Although Alter didn't present any evidence, the slide was blunt: “The data in the Lombardi et al. Science manuscript are extremely strong and likely true, despite the controversy,” one bullet point said. “We (FDA & NIH) have independently confirmed the Lombardi group findings,” read another.
After the editor of a Dutch newsletter discovered the slide on the meeting's Web site and sent out a press release, the CFS world exploded, again. “Is this it, then, the big one? Holy ****!!!!!” one blogger wrote on the Phoenix Rising forum.
Some researchers had doubts. True, Alter had a stellar reputation, but he wasn't a retrovirologist. And Lo in the 1990s had pushed the widely discredited theory that a Mycoplasma infection played an important role in HIV causing AIDS.
Yet news of the impending confirmation had a big impact on a special XMRV panel at the AABB, an international association focused on transfusion medicine. The panel, which had heard about the Lo-Alter study before the news broke, recommended on 18 June that AABB members discourage CFS patients from donating blood.
Patients and scientists alike were eager to see the data. Lo and Alter had written a paper accepted by PNAS, but they ran headfirst into an institutional roadblock: Retrovirologists at the U.S. Centers for Disease Control and Prevention (CDC) had their own paper in preparation that showed no evidence of XMRV antibodies, proteins, or DNA in well-characterized CFS patients. Faced with contradictory results from two teams of researchers under its purview, the U.S. Department of Health and Human Services (HHS) ordered the groups to delay publishing until each could review the other's data.
Patients cried foul. Many had long distrusted the federal government, and especially CDC, for not taking CFS seriously and suppressing research results. Now, they said, CDC was trying to bury another theory it didn't like. The author of a blog, CFS Central, called for aggressive, ACT UP-style protests at blood banks to demand publication: “I believe we need to act quickly, before the FDA/NIH paper is killed.” Some scientists thought the federally ordered delay impinged on scientific freedom. Why not publish the competing papers and let other researchers scrutinize them? “It was very strange business,” Coffin says. Alter says nothing sinister was afoot and that the papers were never in peril. Retrovirology published the CDC study on 1 July 2010, and the Lo-Alter study ran in PNAS on 23 August.
Once they saw it, many researchers concluded that the Lo-Alter data didn't confirm the Lombardi paper at all. They had found that 86.5% of samples from CFS patients harbored DNA from mouse retroviruses as did 6.8% of healthy controls, but it was not XMRV; the sequences were more closely related to a different, well-known group, the murine leukemia viruses (MLVs). It was as if a new suspect suddenly had been nabbed. Miller says the findings actually argued against Lombardi et al. “If XMRV is everywhere and these guys are doing the same experiment, why didn't they find XMRV?” he asks. Alter now says he regrets asserting that the paper confirmed the XMRV results and that it was “naïve” to show the slide in Zagreb. “That is something I shouldn't have said because we really hadn't found it,” he says. “I somehow got to be the spokesperson for this. I had no idea what it would mean.” Lo agrees that the team didn't find the virus that Mikovits and Lombardi had reported.
Mikovits to this day contends that the Lo-Alter paper confirms Lombardi et al. and insists that from the beginning, she viewed XMRV as one of many gammaretroviruses, which includes the MLVs, involved with CFS. In the Lombardi study, some patients tested negative in PCR tests for XMRV and yet produced MLV-related proteins, she claims, but they decided to count them as negatives. She has another serious regret about the paper. “I'd not put XMRV in the title,” Mikovits says. “We never considered that it would be a single sequence.”
As more negative data poured in, Mikovits and Lombardi became ever more ardent. Improved techniques now found the virus in almost every CFS patient, they said.
Patient groups began to see Mikovits as a martyr—a Galileo-like figure fighting an all-powerful scientific establishment to expose the truth. But many of her initial supporters had joined the growing camp of skeptics. “I began comparing Judy Mikovits to Joan of Arc,” Coffin says. “The scientists will burn her at the stake, but her faithful following will have her canonized.”
Mikovits has her own theory about when Coffin changed his mind. She and Lombardi had found evidence, not included in the Science paper, that XMRV was also linked to autism. On 11 November 2009, Lombardi presented those data at a meeting at the Cleveland Clinic. “You don't talk about autism in the U.S.—it's too politically charged,” Mikovits claims Coffin told him. She believes Coffin turned against her that very day. Coffin confirms he was upset that Lombardi presented such preliminary data on such a fraught topic but says, “I did not ‘turn against’ Judy at that or any other point.”
Mikovits further lost credibility with her contribution to an early study organized by the Blood XMRV Scientific Research Working Group, formed by HHS 1 month after the publication of Lombardi et al. It included safety experts such as Susan Stramer of the American Red Cross, as well as Mikovits, Alter, Coffin, and other players in the XMRV saga. The group had devised a four-stage program that included sending blinded samples to various labs to see whether they could detect XMRV. At a meeting on 14 December 2010, the working group discussed the results of the second stage of its study, which again did not support Mikovits's findings. Her lab had found XMRV in a sample from a healthy person who all labs agreed beforehand was negative for the virus.
Mikovits had an explanation. The false positive was caused by a postdoc who mistakenly used the same needle twice to lyse cells and shear DNA, contaminating the sample. She dismisses the error as trivial, the result of working late at night on a weekend because of repeated power failures in a new building, coupled with intense pressure from the working group to get results quickly. “People make mistakes, and we reported it as a mistake,” she says.
But many researchers threw up their hands: How could WPI botch a study essential to the survival of its theory? The University of Washington's Miller was astonished that Mikovits didn't do the critical tests herself and that they were rushed.
Ruscetti is not bothered that most of his peers think he's wrong. “I've been there before,” he says. But he thinks Mikovits has been treated unfairly. “I've been in science for 35 years, and she's as honest a scientist as I've ever met,” he says.
More setbacks followed. A week after the working group meeting, Retrovirology published four devastating papers online that together made a compelling case that a contaminant marred both the Lombardi et al. and Lo-Alter studies. Two reports co-authored by Coffin looked for XMRV and MLV in both CFS and prostate cancer patients; every positive sample they found also harbored mouse DNA, suggesting that a reagent had become contaminated. Another report put a finer point on it: Researchers discovered MLV—at least 97% similar to the Lo-Alter viruses—in a commercially available enzyme used in a PCR kit. Some speculated that perhaps the patient samples were handled more than the controls.
The fourth study, led by Greg Towers at University College London, was even more damning. It was a follow-up to an earlier report, published in the April 2009 issue of the Journal of Virology by Miller's group, that described the discovery of XMRV in a cell line called 22Rv1, which was derived from a human prostate cancer patient. At the time, Miller thought the finding supported the theory that the virus infects humans, as he assumed the original prostate tumor used to make the cell line harbored XMRV. Now Towers's team had compared the genetic sequence of XMRV from different 22Rv1 lines with reported sequences from patients.
The cell line viruses proved ancestral to those in patients. There was also more genetic diversity in viruses from different 22Rv1 lines than different patients, precisely the opposite of what should happen if the virus infected humans: Immune system pressure typically forces a virus to diversify as a means of escaping attack. Towers concluded that XMRV never infected humans and that the cell line virus had somehow contaminated the patient samples.
In March 2011, Coffin and NCI virologist Vinay Pathak delivered what many thought was the final blow to XMRV at the Conference on Retroviruses and Opportunistic Infections in Boston. Working with Oya Cingöz in Coffin's lab, Pathak had analyzed the 22Rv1 cell line all the way back to its origins, a prostate cancer patient at Case Western Reserve University in Cleveland, Ohio. To create the cell line, researchers had used a common technique called passaging in which human tumor cells are grown repeatedly in mice and then harvested. Early versions of the cell line, still stored in university freezers, had no XMRV. But two different strains of mice used in the later experiments each harbored DNA that matched half of the virus. This suggested XMRV was created in the 1990s when two viruses combined in a lab culture, and the widely used cell line spread the virus in many labs, infecting other cell lines, too. The finding furthered doubts that XMRV had ever infected a human being—let alone had a role in either CFS or prostate cancer.
The accidental-origin evidence hasn't convinced Mikovits. “Yes, that can be an origin of an XMRV,” she says. “But it could have arisen multiple times. It's not one sequence.” Pathak contended the chances that the exact same virus would arise twice were the same as dropping a quarter from a helicopter flying over the Grand Canyon and having it land on a quarter on the ground.
“It's all contamination,” Coffin concluded, which outraged Mikovits. “How can John Coffin shut down research like that?” Mikovits shouted during one interview, her blue-gray eyes shooting fire. “He's not God!” She speculates that perhaps the U.S. government, afraid of the huge consequences of a widespread XMRV outbreak, was trying to discredit her work. “We can't afford another public health crisis,” she said.
Eventually, two studies that looked for XMRV in fresh blood samples taken from patients in the Lombardi paper failed to find it. Ila Singh, a virologist at the University of Utah in Salt Lake City, consulted with Mikovits and Lombardi to copy their protocols precisely, and among 100 CFS patients in her study, 14 had tested XMRV-positive by WPI. Singh and co-workers found 5% of both patients and controls to be XMRV-positive—a finding they traced back to a contaminated enzyme used in a PCR reaction.
With help from Peterson, UCSF virologist Jay Levy examined 43 patients who tested XMRV-positive at WPI. Peterson says he wanted to help his former colleagues at the institute. “I went to Jay Levy to prove them right,” he says. All samples tested negative. Again, Mikovits said the study was flawed.
“They didn't do one thing we did,” Mikovits says.
“We did it exactly the way they did it,” Levy says.
On 31 May, Science published the Levy report and the Coffin-Pathak origins study online, along with a so-called Editorial Expression of Concern by Science Editor-in-Chief Bruce Alberts. “The study by Lombardi et al. attracted considerable attention, and its publication in Science has had a far-reaching impact on the community of CFS patients and beyond,” he wrote, before stating that “the validity of the study … is now seriously in question.” “What Science giveth, Science taketh away,” Johnson blogged on Phoenix Rising.
Alberts and Science Executive Editor Monica Bradford had first suggested that Mikovits and her co-authors retract the paper voluntarily. “Science feels it would be in the best interest of the scientific community,” they wrote in a 26 May letter. Mikovits was livid and questioned Alberts's motives. “Who wrote that letter? I don't think it was Science,” she says. The co-authors thought the retraction request was premature, too. “What if we walk away from this based on contamination and it's not contamination?” Lombardi asked. “You've got to give us time to figure this out.”
Alberts stresses that they floated the retraction idea because Science already planned to publish the Expression of Concern. “It wasn't a public call for retraction,” he notes, emphasizing that the recipients shared it with the media. He also does not think it would have been premature, although he says it's often a tough call whether to retract a paper. “Ultimately, it requires expert judgment and a lot of sensitivity to the issues,” he says. “We had lost confidence in the results.”
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CREDIT: KING'S COLLEGE LONDON The growing rift between XMRV believers and doubters became painfully obvious at a June retrovirology meeting in Leuven, Belgium, where the two camps literally kept their distance. In the scientific sessions, questions were mostly polite and informative. During coffee breaks and a poster session with Belgian beer and cheese, Mikovits, her old friend Ruscetti, and De Meirleir, who helped open RedLabs USA, stuck together and barely talked to their scientific opponents. The scientific debate was grinding to a halt.
“I don't care if nobody else in the world wants to work on it!” Mikovits exclaimed at one point, rolling her eyes. “Fine, leave us alone!” When Mikovits's anger subsided, she appeared earnest and even confused by all the criticism. “The virus is real. … I have isolated it from patients. I know it's there,” she said. “Believe me.”
It wasn't just that scientists were growing tired of the debate, Wessely says. Some were put off by the “appalling, unforgivable attacks” by some patient advocates on those who criticized WPI's findings. Wessely says he has received death threats in recent years. “People will rather go over the Niagara in a barrel than ever getting involved in CFS again,” he says.
Silverman notified Science and his collaborators on Lombardi et al. of his discovery that the data he supplied for the paper were wrong. Resequencing samples he tested for the study revealed that somewhere along the line, VP62 had contaminated them. Without casting blame or explaining how the contamination occurred, the partial retraction says two figures and a table that reported viral sequences, including one that showed XMRV in the family tree, were “spurious.”
In late August, the Blood Working Group completed its roughly $500,000 study, which conclusively determined that no one need worry about XMRV or MLVs in the blood supply. The nine labs—which included WPI, Ruscetti, and Lo at FDA—each had received blinded samples from 15 negative controls and 15 others who had tested positive for a gammaretrovirus in Lombardi et al. or Lo-Alter. Different teams cultured the virus, looked for antibodies to it, and used PCR to fish for DNA. All labs could use whatever assays they chose.
Only WPI and Ruscetti found intermittent evidence of viruses or antibodies in patients, but they also reported similar numbers of positive responses in the negative controls. What's more, there was no agreement between the two labs on which patient samples tested positive. “What this says, at the very minimum, is that we can't find it reliably in the blood of patients we found it in before,” Ruscetti says.
Ruscetti understands why many of his peers think Lombardi et al. and Lo-Alter have now run their scientific course. “It is quite legitimate for those people to say maybe these two papers were wrong,” he says. But he emphasizes that many scientific unknowns remain about XMRV. He points to a study in the Journal of Virology in May that intentionally infected macaques with XMRV. It shows that XMRV moved out of the blood of the monkeys but stayed in tissue reservoirs and that antibodies disappeared. “We know nothing about the viral life cycle,” he says.
Blood Working Group member Michael Busch, head of the Blood Systems Research Institute in San Francisco, California, says Ruscetti, Mikovits, Lo, and Alter deserve kudos for participating fully in the study. “I commend them on their scientific integrity and commitment to the scientific process,” Busch says. “This has been a difficult and disappointing process for them and for CFS patients, but hopefully we have all learned lessons that will guide future research and lead to discovery of the cause and cure of this disease.”
The results of yet another multilab study are due early next year. Led by Ian Lipkin of Columbia University, the $2.3 million project plans to test 150 samples from CFS patients and create a repository of their blood so that future putative causes of the disease can more easily be tested. Lipkin says the study will continue despite the Blood Working Group's negative results. “Our study designs are different, our power is different, our subjects are different, and our assays are different,” Lipkin says. “Whether our results will differ remains to be seen.”
Mikovits and Ruscetti are soldiering on. “As long as there are scientific reasons to continue, I think it's incumbent upon me to do it,” Ruscetti says. “I owe it to both the scientific community and the patient community.”
Mikovits says it is irresponsible to dismiss the link between XMRVs and CFS at this point. “Anyone who says this is a lab contaminant has drawn the wrong conclusion and has done a disservice to the public,” she says. Okay, maybe not as many CFS patients have XMRVs as they initially reported, but she's still convinced that a gammaretrovirus is in at least 20% or 30% of them. “I know of hundreds if not thousands of people with evidence of this infection, from what we've done over the last 3 years,” she says. “I don't know what it means. And I'm gong to keep looking for in vivo reservoirs like the ones seen in the macaques, and I'm going to try to figure out mechanisms of pathogenesis, epigenetics, or other things. I'm not going to stop studying it.”
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Robert H. Silverman,
Jaydip Das Gupta,
Vincent C. Lombardi,
Francis W. Ruscetti,
Max A. Pfost,
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Daniel L. Peterson,
Sandra K. Ruscetti,
Rachel K. Bagni,
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Supporting Online Material
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Simone A. Glynn,
Anthony L. Komaroff,
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Leslie H. Tobler,
John Hackett, Jr.,
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Indira K. Hewlett,
Harvey J. Alter,
Jeffrey M. Linnen,
John M. Coffin,
Mary F. Kearney,
Francis W. Ruscetti,
Max A. Pfost,
Jerry A. Holmberg,
Michael P. Busch,
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