of the accompanying comprehensive writeup on the whole XMRV issue,
'False Positive'.
-------------------------------------------------------------------------
Failure to Confirm XMRV/MLVs in the Blood of Patients with Chronic
Fatigue Syndrome: A Multi-Laboratory Study
http://www.mediafire.com/?ce28mj77hc68e2l
False Positive
http://www.mediafire.com/?85zgjb21pecc1zr
Partial Retraction
http://www.mediafire.com/?2t8h57csvgm62t2
---------------------------------------------------------------------------=
-
Science 23 September 2011:
Vol. 333 no. 6050 pp. 1694-1701
DOI: 10.1126/science.333.6050.1694
False Positive
Jon Cohen, Martin Enserink
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.
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.
Think again.
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.
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).
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.
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 =D7 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."
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=B0C 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.
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 "na=EFve" 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
Cing=F6z 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."
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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