Corante

About this Author
DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

Chemistry and Drug Data: Drugbank
Emolecules
ChemSpider
Chempedia Lab
Synthetic Pages
Organic Chemistry Portal
PubChem
Not Voodoo
DailyMed
Druglib
Clinicaltrials.gov

Chemistry and Pharma Blogs:
Org Prep Daily
The Haystack
Kilomentor
A New Merck, Reviewed
Liberal Arts Chemistry
Electron Pusher
All Things Metathesis
C&E News Blogs
Chemiotics II
Chemical Space
Noel O'Blog
In Vivo Blog
Terra Sigilatta
BBSRC/Douglas Kell
ChemBark
Realizations in Biostatistics
Chemjobber
Pharmalot
ChemSpider Blog
Pharmagossip
Med-Chemist
Organic Chem - Education & Industry
Pharma Strategy Blog
No Name No Slogan
Practical Fragments
SimBioSys
The Curious Wavefunction
Natural Product Man
Fragment Literature
Chemistry World Blog
Synthetic Nature
Chemistry Blog
Synthesizing Ideas
Business|Bytes|Genes|Molecules
Eye on FDA
Chemical Forums
Depth-First
Symyx Blog
Sceptical Chymist
Lamentations on Chemistry
Computational Organic Chemistry
Mining Drugs
Henry Rzepa


Science Blogs and News:
Bad Science
The Loom
Uncertain Principles
Fierce Biotech
Blogs for Industry
Omics! Omics!
Young Female Scientist
Notional Slurry
Nobel Intent
SciTech Daily
Science Blog
FuturePundit
Aetiology
Gene Expression (I)
Gene Expression (II)
Sciencebase
Pharyngula
Adventures in Ethics and Science
Transterrestrial Musings
Slashdot Science
Cosmic Variance
Biology News Net


Medical Blogs
DB's Medical Rants
Science-Based Medicine
GruntDoc
Respectful Insolence
Diabetes Mine


Economics and Business
Marginal Revolution
The Volokh Conspiracy
Knowledge Problem


Politics / Current Events
Virginia Postrel
Instapundit
Belmont Club
Mickey Kaus


Belles Lettres
Uncouth Reflections
Arts and Letters Daily
In the Pipeline: Don't miss Derek Lowe's excellent commentary on drug discovery and the pharma industry in general at In the Pipeline

In the Pipeline

« Has Luc Montagnier Lost It? | Main | The Life of a Paper »

January 11, 2011

XMRV: It's Ugly, But That's Science

Email This Entry

Posted by Derek

How's the XMRV / chronic fatigue syndrome connection holding up? Not real well. Science has a roundup of the latest news in the area, and none of it looks encouraging. There are four studies that have come out in the journal Retrovirology that strongly suggest that earlier positive test results for the virus in CFS samples are just artifacts.

For one thing, when you look closely, it turns out that the sequences from cell-cultured XMRV samples are quite a bit more diverse than the ones taken from widely separated patients at different times. And that's just not right for an infectious agent; it's the opposite of what you should see. A number of supposedly XMRV-specific primers that have been used in such assays also appear to amplify other murine viral sequences as well, and samples that show positive for XMRV also appear to have some mouse DNA in them. Finally, there's reason to believe that some common sources of PCR reagents may have murine viral contaminants that blow up this particular assay.

Taken together, these latest results really have to make you cautious in assigning any role at all to XMRV based on the published data. You can't be sure that any of the numbers are what they're supposed to be, and the most parsimonious explanation is that the whole thing has been a mistake. To illustrate the state of things, you may remember an effort to have several labs (on both sides of the issue) test the same set of samples. Well, according to Science. . .

Some had hoped that a project in which several U.S. labs are testing for XMRV in the same samples would clear up the picture. But so far this effort has been inconclusive. Four CFS patients' blood initially tested positive for XMRV at WPI and the U.S. Centers for Disease Control and Prevention but not at an NCI lab. When all three labs tested new samples from the same patients, none found XMRV—for reasons that aren't yet clear, says Coffin. The group now plans to test blood from several dozen CFS patients and controls.

No, this isn't looking good at all. It's pretty typical, though, of how things are out at the frontiers in this business. There are always more variables than you think, and more reasons to be wrong than you've counted. A theory doesn't hold up until everyone who wants to has had a chance to take some big piñata-shattering swings at it, with weapons of their choice. So, to people outside of research: you're not seeing evidence of bad faith, conspiracy, or stupidity here. You're seeing exactly how science gets done. It isn't pretty, but it gets results in the end. Circumspice.

Comments (70) + TrackBacks (0) | Category: Analytical Chemistry | Infectious Diseases


COMMENTS

1. Anonymous on January 11, 2011 10:32 AM writes...

I guess you are not so updated with the "other" side of the coin, I reccomend you have a look on the studies that did find XMRV and the different than PCR methods they used, and the measures they took to avoid contamination.

Example: FDA Advisory Meeting last December:

DR. ALTER:

"Since Dr. Lo had to leave early, I felt I had to come up and do some defense of him and Judy as well. I think, when a group finds a new agent, they become biased that this agent is real. When another group doesn't find an agent, they become, I think, even more biased that the agent is not real. That leads to this kind of contentiousness.

I think our goal should be not to bring the other side down, but to find the truth. I think the truth will out over the next year, with studies that are already planned.

At this point I concur that we have no evidence for causality. That's going to be very difficult to come by, especially when we are detecting at the limits of detectability and when assay performance is very critical to get equal results.

But I still want to counter by saying I think the current evidence for disease association is very strong, even though not universally confirmed. But it has been confirmed now in at least four studies, two of which were presented today, that either XMRV or a polytropic MLV is associated strongly with chronic fatigue syndrome. A point that I think was misrepresented today: In those labs who do find the agent, it is very reproducible. Judy has found the same patients to be positive by culture year after year. We have found a patient to come back after 15 years and still be positive. So this is not a single, isolated finding. It's confirmed by sequencing. It's reproducible over time.

Dr. Hanson has shown today how critical the assays are. When she tweaked her assay, she went from no findings to findings almost identical to the Lo lab. The diversity is now being confirmed also in the original WPI group. XMRV isn't the only agent even in the WPI lab.

Despite the very legitimate concern for contamination -- I think this is a serious issue -- there have been hundreds of negative controls in the same laboratory that are always consistently negative. An extremely sensitive mouse mitochondrial DNA has always been negative in the Lo laboratory. Lo has done the [IAP] assay that Dr. Coffin recommended. That is also negative. There just has been no evidence for contamination. Although you could say maybe the negatives could be negative somehow and the positives positive for contamination reasons, it really is not logical that that would be so.

I'm not a molecular biologist. I defer to Dr. Stoye, who is world-renowned in that area. But just as a simple doctor, it seems to me that you have used single-case anecdotal evidence to knock down the various possibilities. I just want to make a case to the committee that you can't -- your conclusion is that anything can happen in assays, and therefore it probably has happened this time. I think using that kind of anecdotal probability is not valid to negate reproducible data from four different laboratories. So at least keep that in mind.

Lastly, I'm not a chronic fatigue doctor, but I have learned a lot about chronic fatigue in the last six months and have spoken to a lot of patients. I'm absolutely convinced that when you define this disease by proper criteria, this is a very serious and significant medical disease, and not a psychological disease. It has the characteristics of a viral disease. It usually starts with a viral-like illness. If XMRV is not the causative agent -- and it may well not be -- there is still need by other groups to look for the next agent which may be the case"

Source: http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/BloodProductsAdvisoryCommittee/ucm239304.htm

Permalink to Comment

2. SonicChronic on January 11, 2011 10:38 AM writes...

I wonder if the homogenity in human titers is representative of some sort of selection process where only a certain sequence can avoid an effective immune challenge. I don't know anything about virology but am just sayin.....

Permalink to Comment

3. Anonymous on January 11, 2011 10:57 AM writes...

Any serious PCR expert could look at the initial Science paper and in 3 minutes know that it was PCR contamination combined with an insufficiently specific PCR assay. I laughed when I saw it.

Permalink to Comment

4. xand on January 11, 2011 11:55 AM writes...

Any serious comment should have known that the Science paper did use 4 different methods for detecting XMRV, and never a simple PCR as you suggest, they cultured the virus for 45 days, they found antibodies and an immune response... this virus is NOT contamination.

DR. MIKOVITS:

"These patients in the U.K. have not [had virologic/infectious disease workups]. It is a psychosomatic disease in the U.K., and they can't get those types of medical treatments easily and maintain their benefits. In our study in Science, the answer is yes. These patients have multiple chronic active infections: EBV, HHV-6, CMV, shingles, mycoplasma as I mentioned, We see everything .

It looks to us like an AIDS patient, with an obvious hypothesis being that the retrovirus causes the underlying immune deficiency. But it alone can't cause the disease. It needs the co-pathogens. You can have HIV without having AIDS, but you can't have AIDS without having HIV plus one of 25-odd co-pathogens."

Source: http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/BloodProductsAdvisoryCommittee/ucm239304.htm

Permalink to Comment

5. AKS on January 11, 2011 12:06 PM writes...

Somewhat generally related to this, but have you seen this interesting article in the New Yorker:

http://www.newyorker.com/reporting/2010/12/13/101213fa_fact_lehrer?currentPage=all

Might make for an interesting blog post and discussion here.

Permalink to Comment

6. Edugreat on January 11, 2011 12:17 PM writes...

Below is part of a paper published on the WPI website in response to the four "negative" papers.
full paper at this link: http://www.wpinstitute.org/news/news_current.html

The main question regarding this issue of "contamination" is how does it explain the integration into the DNA of cancer tissue and the development of an antibody immune response. How can my wife's body have developed antibodies to a lab contaminate 500 miles away?!!!
-----------
XMRV: A Human Retrovirus with Unknown Pathogenic Potential, Not a Lab Contaminant

The recent proclamation that “XMRV is not the cause of CFS,” came from an individual who did
laboratory experiments to show how PCR experiments can become contaminated. These results
have nothing to do with the reality of a disease or the methods used by those who have detected
XMRV in the blood and tissue of patients found to be infected. The positive studies, which
cannot be explained away by PCR experiments, are those which have used multiple methods to
show that XMRV is a live replicating gamma retrovirus in human blood and tissue samples using
the gold standard methods of viral isolation and antibody testing, in addition to PCR.
Unsupported conclusions, such as the one offered by the Wellcome Trust spokesman, often
create sensational headlines but do little to move science forward. Authors of the positive
XMRV studies have been extremely careful not to claim causality, realizing that more scientific
research is required to make such a statement. However, one fact still remains clear. Not one of
the negative studies changes the results of the scientific research done by Lombardi et al., Lo et
al., Urisman et al., and Schlaberg et al.
The WPI-led scientific study, which rigorously ruled out contamination, revealed high
associations of gamma retroviruses with physician-diagnosed CFS patients, using four different
methods of detection. Recent commentary associated with the negative research papers on
XMRV, which used only one testing method, claimed that these studies proved that XMRV was
not the cause of human disease. On the contrary, what the authors of the “contamination studies”
confirmed is something that most experienced scientists already know; there are risks associated
with using PCR if one does not properly control for contamination. They cannot conclude that
other research groups had the same problems or that “XMRV is not the cause of CFS”.
Most significantly, the recent Retrovirology publications failed to address the most
important pieces of scientific evidence of human infection in the previous XMRV studies,
including the fact that XMRV positive patients produce human antibodies to gamma
retroviruses, XMRV integrates into human tissues, and infectious virus has been cultured
from the blood of hundreds of patients with a diagnosis of Chronic Fatigue Syndrome and
M.E. Humans do not make antibody responses to mouse DNA sequences from
contaminated lab experiments. The Retrovirology studies only point out that XMRV
research cannot be done in a mouse laboratory without extreme caution and should not
rely solely on PCR methods.

Permalink to Comment

7. ME/CFS since 2010 on January 11, 2011 1:05 PM writes...

Back in May 5th 2010 i was sexually involved with a girl and when we were doing it i noticed the condom had broken when i withdrew my first thought was "i hope i don't get this girl pregnant" but she told me she was on the pill so i went on with my life,(all these following symptoms i have never seen or felt before in my life) two weeks went by and i had a couple of hot flashes but didn't make a big deal of it, two more weeks went by and i noticed i had many many little pimples on my back right behind my armpits around this same time i started to feel confused but i thought maybe i didn't get enough sleep, around the 5th week i started to get worried because i was feeling nauseated and somewhere around the 6th week i had a "viral spike" i got really sick with all sorts of symptoms, nausea,confusion,weakness, constipation,fatigue, night sweats, lost like 15lbs in a few days, i had a really bad rash on my back.

i thought for sure i had gotten HIV, i went to get tested for HIV on July 21, negative.

i thought mm maybe it's just a stomach flu and it will go away, but the symptoms wouldn't go away if anything i felt like i was getting worse, i kept testing for HIV at many different places, i went to different cities to different health clinics to get tested like every week because the counselor at my local health clinic kept telling me i couldn't get tested anymore because it was negative this went on for about 4 months( by this time i had already been to ER twice because of all the symptoms had been to GI specialist, dermatologist,my primary MD, two infectious disease guys and many different health clinics).

i was sure i had gotten some weird HIV strain that the current test weren't able to pick up, and the funny thing is that all new counselors and doctors i went to see kept suggesting i tested for HIV because my symptoms mimic HIV i told them i had already tested negative and they would say then it's something else.
then i went to the website thebody.com and asked doctor Bob if i had gotten HIV and why my tests kept coming back negative, he suggested i went to see an internist, i went to see an internist at the Northwestern Memorial Hospital in Chicago, i told him what was going on and gave him my binder with all my medical records from the previous months and gave him all my negative HIV results papers, since i had already gotten done head scans,abdomen scans,many CBCs,heart ultrasounds, full panels of stds, he suggested i get a brain MRI(because i kept complaining of weird feelings in my head like it was shrinking and like it was burning and something in there was moving around) he ordered a bunch of test, liver function,kidney function, CBC, HIV 1/2/O and some others and the only thing that was abnormal was the inflammation test it showed that i had inflammation somewhere in the body, i told him it was in the brain i could feel it.

The brain MRI results came back and this was my first big break it showed brain demyelination, i kept telling my doctor that this happens with a viral invasion to the brain, and he said, but what virus, your blood counts are perfect and i can't find this virus, so i suggested he tested me for HTLV 1/2 and it was negative,he sent me to a neurologist she was booked like a month in advance, so in the mean time with no more doctor visits and started to read about XMRV, and at the same time kept testing for HIV at the RUSH university and kept coming back negative, finally i went to see the neurologist, and the very first possible diagnostic was MS, and then i remembered that the girl i was with had told me she was a CFS(later on was diagnosed as atypical MS) sufferer since 2004 and she's always very really tired and faints and has seizures sometimes by then i had read somewhere that XMRV was being linked to CFS, then i had no doubt i had gotten something infectious, the neurologist ordered a spine MRI to confirm MS but it came back as normal and she decided to sent me to another neurologist(i kept calling her and leaving her messages about XMRV and she never responded) i decided instead to go see one of the top infectious disease guys at the Northwestern Memorial Hospital and told her what was going on, she saw my symptoms and she said have you gotten tested for HIV i said yes it's negative, how about HTLV she asked i said yes negative, i told her about the new XMRV virus discovered and was now being linked to CFS and i told her the girl i was with had CFS for 6 years and she said i think you should go see the internist again and have him set you up with a counselor, i was then full with rage i mean who in their right mind would think that having brain demyelination is a mental thing.

My current symptoms are, progressive brain demyelination, major GI problems, i have developed food allergies i can't eat meat anymore makes me puke, extreme fatigue,multiple eruptive dermatofibromas,extreme lower back pain, major cognitive problems, confusion all the time,hands and feet numbness, daily headaches, pain in my spleen, swollen tongue with teethmarks around it, white thrush, pain in my liver and pancreas i have also developed an infection in my urinary tract, pain in my testicles, tingling in my eyes all the time, brain fog and brain inflammation and i feel as if i had a bunch of aunts running around in my brain,(note that i had never in my life seen or felt these before, i didn't even knew what brain demyelination was, i never thought something like that could possibly happen to a human being) and i tested again a month ago for HIV and HTLV, CMV,candida,herpes 6,(just to make sure that wasn't it) and was all negative.

i am really having a hard time understanding how some people think XMRV might not exist i mean isn't the evidence obvious, all they have to do is look at the history of CFS and see how they were these clusters, meaning there's a pathogen infecting people, look at the brain MRI for god sakes, they look like aids MRI but yet these people don't have aids, really the most logical reason for somebody to have a swollen tongue is because you have a virus in your blood that your spleen is trying to clean, white thrush means you have a compromised immune system and something is killing your good bacteria seriously it does not take a genius to figure these things out, it's amazing how some doctors, infectious disease guys,immunologist, virologist have studied for years and years and have spent thousands of dollars in education and yet they keep missing the most obvious things, i mean 30 years and billions of dollars have been spent in HIV research and these brilliant minds haven't figured out that there's something else out there that causes aids like illness and aids like brain MRIs, my five year old nephew could figure this one out really, and to think that some of these brilliant minds think that XMRV a retrovirus might not cause disease, really? when in history has a retrovirus not shown to cause disease when? i wanna know because it seems to be a big puzzle that some people can't figure out, a retrovirus isn't and will never be benign, anyways i just thought i would share this story with you guys because it's really fresh and i still remember every detail like it happened, most CFS and have been sick for years and don't remember what is like to be healthy but i do, 8 months ago i could go to the gym and run 5 miles nonstop, now i can't even get to my third floor condo with out feeling like my heart is gonna blow and nothing in my life is what it used to be this is just a horrible hell.

I really hope that people at the CDC would find it in their hearts to put all politics aside and admit that XMRV is a very dangerous pathogen not only because there are millions of people infected already but because millions more will get infected as well.

The blood supply has been contaminated with XMRV with no doubt and people will get sick from it for sure, when i thought i had gotten HIV i was at both HIV forums poz.com and thebody.com and many many people that had the "worried well" label kept testing negative for HIV for months some even have been in these forums for a couple of years, they reason they kept testing for HIV is because they thought they had gotten HIV exactly like my case specially because they had many many symptoms after having unprotected sex, it turns out that a lot of them were going the Red Cross to donate blood because they thought that Red Cross was screening for all transmissible diseases and it turns out that most of them had their donations approved and later on many many have already tested positive for XMRV and who knows how long this has been going on for.

Permalink to Comment

8. RRM on January 11, 2011 1:33 PM writes...

Good write up. An additional, revealing piece of information that is not in the report by Science:

In the first round of testing for the "project" (for the Blood Working Group), when two labs found XMRV, the blood was also collected by the scientists that had reported an asociation with XMRV.

For the second round of testing, an independent phlebotomist visited the patients to collect new blood samples, and then all three labs found no XMRV.

Permalink to Comment

9. courtney on January 11, 2011 1:39 PM writes...

ME/CFS since 2010, I feel like it could beneficial for you to consider checking out the HHV-6 foundation (www.hhv-6foundation.org) and consider emailing info@hhv-6foundation.org to get some feedback on your situation. They are incredibly knowledgeable on many of the viruses that result in MS-like lesions (one of them being HHV-6).

Permalink to Comment

10. carlos on January 11, 2011 5:56 PM writes...

RRM...where did you get that information? That is actually relevant, 2 possible lectures:
a)WPI brought contamination in the samples. (although that would not explain that one of the 3 labs was not able to find XMRV)
b)The "independent collector" is not so independent. (But then again they would call me stupid for thinking on conspiracy)
Things are definitly not clear.

Permalink to Comment

11. aidan walsh on January 11, 2011 7:07 PM writes...

i will be very very glad when they make up their minds if it is or it is not xmrv. i read an article recently in huffington post about a percentage of cfs patients testing positive to giardia and the same work was replicated by a huston team. i think it was dr. leo galland and he said an epidemic of cfs in california actually followed a giardia outbreak and if not detected in patients can persists from weeks to years. it is found by stool samples in specialty labs and is curable with flagyl and or other meds. a funny thing with this small intestine parasite is people cannot tolerate antacid medicines, it actually makes them worse. funny thing now is i get extremely ill when they give me calcium d3 and my stomache gets very bad.i believe they are a type of antacids. could be something to this theory. in the meantime i will stick to www.waterure.com www.watercure2.org until these genius scientists make up their minds why we are all slowly dieing....aidan walsh southampton, u.k. god bless all the cfids sufferers... get well soon...cure/peace in 2011....

Permalink to Comment

12. urbantravels on January 11, 2011 8:37 PM writes...

Where I see evidence of bad faith is not in the published science itself, but in the misrepresentation of the conclusiveness of any one piece of evidence by the researchers themselves, and the subsequent echoing and amplification of those misrepresentations by the press and the Internet.

Such, I believe, was the case with the press release issued by the Wellcome Trust, http://www.wellcome.ac.uk/News/Media-office/Press-releases/2011/WTX064078.htm, which went so staggeringly far beyond the hypothesis presented (and so far unverified by other workers) in the Hue paper (http://www.retrovirology.com/content/7/1/111). To paraphrase Mark Twain, reports of the death of the XMRV/CFS hypothesis have been greatly exaggerated. It would help if the scientists themselves were not so eager to help write the premature obituaries.

Early science is messy and contradictory as hell and can get ugly; scientists, and even some informed laypeople, understand this, and can even see rigorous debate as a good and healthy sign. When information about the messy goings-on get filtered to the outside world via the press, however, either the mess is made to seem like mistakes, confusion and lack of progress (which we ought not to be wasting money on), or the mess is de-emphasized in favor of tidy “conclusions� that are often supported by privileging the claims of one side in a debate that may have multiple sides. (And if conclusions have been reached, we shouldn’t be wasting money on further investigation.)

We have seen how eagerly and uncritically the Wellcome Trust press release was carbon-copied into dozens of press outlets, blogs, Tweets, and spread around the world unchallenged. Why is this a concern, in the particular area of ME/CFS research? Because reporting premature conclusions in this matter serves to reinforce an old and very damaging narrative about CFS: that the cause is impossible to find; that repeated efforts to find the cause have all been blind alleys; that ME/CFS is a ‘vague and ill-defined’ illness, with the implication that an organic cause can never be found – and then we’re back in the “hysteria� wastebasket, with the century-old ideas of Charcot and Freud to weigh us down.

Public opinion affects support for research in very real terms; so does the official stance of the public health agencies. We have already seen several decades in which the NIH officially considered ME/CFS to be a form of depression or conversion disorder - based upon no particular hard evidence - and instructed the press to treat it accordingly. So there were few to protest when ME/CFS research was underfunded, or when funds intended for biological research were diverted elsewhere, or when grant applications for what little money was available were routinely turned down by ‘expert’ panels with no expertise in the disease. The NIH is showing encouraging signs of changing course in recent years, but we have yet to see the result in terms of hard dollars: is there any other disease as serious and disabling as ME/CFS that recieves so little NIH funding?

http://report.nih.gov/rcdc/categories/#bpopup

The recent article that appeared in Science magazine, though reasonably fair overall, ended on a rather sour note, which seems to me a pretty clear indication of how negative perceptions inside and outside the research community can start to lead to a chilling effect on research:

http://www.sciencemag.org/content/331/6013/17.full

“As the new [NIAID/Lipkin XMRV] study gets started, some wonder whether it’s worth the $1.3 million it will cost. Jonathan Stoye of the MRC National Institute for Medical Research in London concedes that the Towers study was “over-hyped.� But he says “it’s pointing people in a certain direction,� away from chasing an elusive link to XMRV. Still, he says, a larger study may be the only way to satisfy [CFS] patients.�

The journalist’s favorite gang of straw men, we all know, is “some.� Who are these “some� people who are already suggesting that $1.3 million is too handsome a sum to spend to get good answers on XMRV, in the face of these supposedly damning recent publications on contamination? Stoye seems to be saying that the only reason to proceed with the study, in his opinion, is to shut up the patients. This implication does a disservice to the NIAID’s motives in ordering and funding the study. If XMRV truly does cause or contribute to human disease, then it’s not just the problem of a group of querulous patients who wants answers – it’s everybody’s problem, and NIH doing due diligence to look for those answers is the proper way to serve the public interest. To suggest otherwise does, indeed, smack to me of bad faith.

Permalink to Comment

13. Warbler512 on January 12, 2011 2:07 AM writes...

OK - Dr. Raciniello proclaimed XMRV done himself, then re-read the four papers, seemed to agree some were overreaching, got the Tribune to retract his worldwide statement, now links his readers to this. Perhaps with a good heart, and Dr. Lowe is welcome to differ, or not. I'm not concerned about Rancaniello's e-mail box melting. I do wonder if a dean at Columbia called him in for a chat about his tenure.

Much as Walter Gunn, the only ally ME/CFS had at CDC in the '80s, was nudged into retirement. Much as John Kerr mysteriously withdrew from one of the negative U.K. XMRV studies, under "academic pressure" as has been put. Kerr's colleague in that study, John Stoye, now NIH spokesman for XMRV (like there's no suitable American) won't disclose why that study never mentioned the samples they received from the U.S., or tell Americans whether their blood was even tested.

Many of us are zoo-fulls of the same pathogens HIV patients get; our blood should have been banned long ago, retrovirus or not. Nonetheless, overlooking Lipkin's shoulder is the same Dr. Fauci who threw and kept ME/CFS under a bus for 25+ years. Patent #WO9205760, the DeFritas retrovirus particles, somehow escaped his attention. NIH didn't just start rejecting grant proposals from WPI either; they've done so since Oct 09, possibly before, as adamantly as in the '80s but for a select few who stayed on-message. CDC, meanwhile, trustworthy Bllod Working Group team-player that they are, keeps producing "results" out of their Atlanta CFS team regarding stress, child abuse, whatever. And we just had to be reminded by some Belgian geniuses in Psychotherapy and Psychosomatics that we "don't live in a vacuum." (We're so stress-maladapted that ARVs wouldn't cure us in any event - just can't read between the lines there.)

From the outside, we may look overly biased, angry, simply unwilling to face "results" that don't suit us. If so, we're not that different from many of our accusers. We're all too aware that scientists don't live in a vacuum either. I don't know what XMRV will turn out to be (is that '06 German respiratory study invalid too?), but I wish a lot more of what I'm watching WAS a conspiracy theory.

Permalink to Comment

14. Justin Reilly on January 12, 2011 3:00 AM writes...

I am not a scientist, but I have read up on the studies. They don't prove that ME is not caused by XMRV as the press release said. And they don't "strongly suggest" contamination. Some very anecdotal reports of unpublished results and remarks by Rein (?) at NCI and Coffin give me a little bit of pause, particularly Coffin's report of finding XMRV in cereal boxes (you heard me!), but I think that the published data, taken as a whole (particularly the DNA integration site in prostate cancer and the antibody response) strongly suggest that XMRV is strongly associated with ME. It is possible that XMRV isn't a major cause of ME. Dr. Rein thinks it doesn't cause prostate cancer because to be consistent with known viral oncogenesis it would have to be present in every cancer cell and he could find it only in a small percentage or not at all- i forget which.

Anyway, Dr. Alter's statement makes great points and should be read by everyone (along with WPI's and Racianello's blogs and podcasts) before deciding what they think. I think the circumstantial evidence is very strong that this disease is caused by one or more retroviruses, whether XMRV itself is a major cause has yet to be determined.

You say "So, to people outside of research: you're not seeing evidence of bad faith, conspiracy, or stupidity here. You're seeing exactly how science gets done. It isn't pretty, but it gets results in the end." Well, with due respect, patients and ME researchers and clinicians are the experts on whether there is bad faith, conspiracy or stupidity, not you. And let me tell you: there is. What about the statement in the press release that "XMRV does not cause ME" strikes you as not being in bad faith? Such distortion of the science and lying is par for the course in CDC, NIH and the UK govt's quarter century war on ME science and patients.

Permalink to Comment

15. Jan on January 12, 2011 3:06 AM writes...

Also please note that the CDC study was not done on patients diagnosed with CFS by their physicians. The CDC's participants were decided to have CFS by the Empirical criteria, which judging by the CDC's own research, is not a useful criteria because they have to make comments like "matching was not maintained" between cases and controls (and since research consistently shows a long-term recovery rate of less than 10%, this is not a problem they should be having).

Jason has shown that with the introduction of the Empirical criteria, the incidence of "CFS" has increased tenfold to coincidentally approximate the incidence of mood disorders, and that, for instance, 38% of those with major depressive disorder, can be classified as having CFS by using the Empirical criteria.

Jason LA, Najar N, Porter N, Reh C. "Evaluating the Centers for Disease Control's Empirical Chronic Fatigue Syndrome Case Definition". J Disability Policy Studies. September 2009 vol. 20 no. 2 93-100. full text available at www.cfids-cab.org/MESA/Jason-10.pdf

The CFS inclusion criteria problem is not limited to the Empirical inclusion.

Merz S. [Chronic fatigue syndrome. More and more differential diagnoses suggest a new view of this syndrome]. Lakartidningen. 2002 Aug 22;99(34):3282-7. PMID: 12362846
"Several CFS definitions have been developed over the years, and it is common for investigators to erroneously compare studies based on different definitions, which nevertheless all use the term CFS. Much of our 'understanding' of CFS does not apply to the small group of patients who fulfill the current (1994) CDC definition."

Jason LA, Helgerson J, Torres-Harding SR, et al. “Variability in diagnostic criteria for chronic fatigue syndrome may result in substantial differences in patterns of symptoms and disability.� Eval Health Prof. 2003 Mar;26(1):3-22. PMID: 12629919
"Chronic fatigue syndrome (CFS) is an illness that involves severe, prolonged exhaustion as well as neurologic, immunologic, and endocrine system pathology... The current investigation examined differences between CFS as defined by Fukuda and colleagues and a set of criteria that has been stipulated for myalgic encephalomyelitis (ME). Dependent measures included psychiatric comorbidity, symptom frequency, symptom severity, and functional impairment. The ME and Fukuda et al. (1994) CFS criteria were compared with a group having chronic fatigue due to psychiatric reasons. Significant differences occurred primarily with neurologic, neuropsychiatric, fatigue/weakness, and rheumatological symptoms. These findings suggest that it might be inappropriate to synthesize results from studies of this illness that use different definitions to select study populations."

Although researchers have suggested this is inappropriate, it is standard to compare results from various inclusions when discussing consistency of results (such as XMRV) and even treatments. Although these differences of inclusion are not apparent to most journalists (and even most medical doctors), the problems caused should be easy enough to understand once they have been brought to people's attention.

The Lombardi paper used Canadian criteria for ME/CFS requiring neurological and immunological signs and symptoms, as well as Fukuda (patients meeting Canadian also meet Fukuda).

The CDC paper on XMRV (Switzer et al.) used Empirical criteria and criticized neurological and immunological signs and symptoms, including a diagnostic-supportive symptom of Fukuda and other noted symptoms of Fukuda (the CDC's official definition), saying they might indicate some disease other than CFS.

The patients the term CFS was coined for had neurological and immunological signs and symptoms:
Buchwald D, Cheney PR, Peterson DL, et al. "A chronic illness characterized by fatigue,
neurologic and immunologic disorders, and active human herpesvirus type 6 infection." Ann Intern Med. 1992 Jan 15;116(2):103-13. PMID: 1309285 http://www.ncbi.nlm.nih.gov/pubmed/1309285

The problem with interpreting and reporting on CFS research is that this whole field is unfortunately highly politicised, to the great detriment of the patients.

See also:
http://myalgic-encephalomyelitis.com/definitions_criteria_protocols.html (links to multiple definitions including the ones discussed; Empirical is listed as "CDC revised," a confusing label since that designation is often used in the literature for Fukuda, which was revised from Holmes, the first CDC definition)

Jason et al. “Politics, Science, and the Emergence of a New Disease: The Case of CFS� Am Psychol. 1997 Sep;52(9):973-83. PMID: 9301342 : http://www.cfs-news.org/jason.htm

Editorial. “Frustrating survey of chronic fatigue syndrome.� Lancet 1996; 348:971. http://www.thelancet.com/journals/lancet/article/PIIS0140-6736%2805%2964917-3/fulltext

Schweitzer M. "Problems continue with U.S. government agencies." 2000. http://www.cfids-me.org/marys/nihprobs.html

Hooper M. "Myalgic encephalomyelitis: a review with emphasis on key findings in biomedical research." J Clin Pathol. 2007 May; 60(5): 466-471. PMC1994528 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1994528/

Carruthers et al. "Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Clinical Working Case Definition, Diagnostic and Treatment Protocols" J CFS, Vol. 11(1) 2003, pp. 7-115 (pp 46-49). http://cfids-cab.org/MESA/ccpc-1.html

For well-replicated biological pathologies, see:

Fletcher MA, Zeng XR, Maher K, et al. "Biomarkers in chronic fatigue syndrome: evaluation of natural killer cell function and dipeptidyl peptidase IV/CD26." PLoS One. 2010 May 25;5(5):e10817. PMID: 20520837

Jammes Y, Steinberg JG, et al. "Chronic fatigue syndrome combines increased exercise-induced oxidative stress and reduced cytokine and Hsp responses," J Intern Med. 2009 Aug;266(2):196-206. PMID: 19457057

Cook DB, Lange G, DeLuca J, Natelson BH. "Relationship of brain MRI abnormalities and physical functional status in chronic fatigue syndrome." Int J Neurosci. 2001 Mar;107(1-2):1-6. PMID: 11328679

Also see:

Light AR, et al, "Moderate Exercise Increases Expression for Sensory, Adrenergic, and Immune Genes in Chronic Fatigue Syndrome Patients But Not in Normal Subjects," J Pain, 2009 Oct;10(10):1099-112. Epub 2009 Jul 31. PMID: 19647494

Permalink to Comment

16. Jan on January 12, 2011 3:22 AM writes...

evidently I missed something; that the CDC was using the wrong patients cannot account for the varied finding with the 4 patients in the Blood Work Group, but n=4 is not a sufficient amount of data to draw any conclusions with. One of those patients, for example, had begun taking medication which could reduce the amount of virus in the blood. Some researcher also hypothesize that the viral load in the blood may fluctuate with time. As you said, there are a lot of variables. These variables may or may not be related to the presence or absence of XMRV in these 4 patients.

And as I noted, there's also a huge amount of politicization, which is vastly detrimental to the funding and progress of ME/CFS research and to the attitude towards and treatments available for these patients.

Permalink to Comment

17. RRM on January 12, 2011 7:02 AM writes...

I have to say that quote of Dr. Alter does seem really strange. For instance:

"when a group finds a new agent, they become biased that this agent is real. When another group doesn't find an agent, they become, I think, even more biased that the agent is not real"

Are there many real-life examples where whole groups of scientists were biased agianst a new finding that turned out to be correct? I know there are uncountable examples of scientists that were biased in favor of findings that turned out to be incorrect. Ironically, there seems to be a clear bias in Alter's observation, as the few "successes" (e.g. Helicobacter pylori) are always better remembered than all those "failures" (e.g. Alter and Lo once also "detected" mycoplasmas in patients with AIDS, a finding that has even been "confirmed" by Nobel laureate Luc Montagnier).

"At this point I concur that we have no evidence for causality. That's going to be very difficult to come by, especially when we are detecting at the limits of detectability and when assay performance is very critical to get equal results."

This is really, really strange. His/Lo's own study was not at all detecting MLV related sequences at the limits of detection. It used one of the least sensitive assays of all studies so far and they did PCR only. The Science authors also didn't initially report any problems to detect the virus.

"A point that I think was misrepresented today: In those labs who do find the agent, it is very reproducible."

It apparently is very reproducable in a non-blinded setting and it is at the limit of detection. I am sure I've read that before, and it usually is not a good sign.

"Judy has found the same patients to be positive by culture year after year."

This statement was made like 14 months after the initial study was published. Also, that is exactly what was investigated in Phase IIb of the Blood Working Group: That same Judy (Mikovits) was free to chose her favorite 4 of the patients that were positive "year after year", then they mixed them up with a healthy control, and after that she couldn't tell these self chosen XMRV infected patients from the healthy, pedigreed negative patient (that was also self chosen and thus one of the patients that was "always consistently negative" in a non-blinded setting).

Admittably too little samples were used to draw definite conclusions from these results, but sure they were unlucky.

"We have found a patient to come back after 15 years and still be positive"
How does that argue against contamination in the slightest? Mix this patient up with 10 healthy and pedigreed negative controls (make sure the blood collection, storage and processing is exactly the same for all patients) and see if you can identify him from those samples.

Permalink to Comment

18. RRM on January 12, 2011 7:02 AM writes...

I have to say that quote of Dr. Alter does seem really strange. For instance:

"when a group finds a new agent, they become biased that this agent is real. When another group doesn't find an agent, they become, I think, even more biased that the agent is not real"

Are there many real-life examples where whole groups of scientists were biased agianst a new finding that turned out to be correct? I know there are uncountable examples of scientists that were biased in favor of findings that turned out to be incorrect. Ironically, there seems to be a clear bias in Alter's observation, as the few "successes" (e.g. Helicobacter pylori) are always better remembered than all those "failures" (e.g. Alter and Lo once also "detected" mycoplasmas in patients with AIDS, a finding that has even been "confirmed" by Nobel laureate Luc Montagnier).

"At this point I concur that we have no evidence for causality. That's going to be very difficult to come by, especially when we are detecting at the limits of detectability and when assay performance is very critical to get equal results."

This is really, really strange. His/Lo's own study was not at all detecting MLV related sequences at the limits of detection. It used one of the least sensitive assays of all studies so far and they did PCR only. The Science authors also didn't initially report any problems to detect the virus.

"A point that I think was misrepresented today: In those labs who do find the agent, it is very reproducible."

It apparently is very reproducable in a non-blinded setting and it is at the limit of detection. I am sure I've read that before, and it usually is not a good sign.

"Judy has found the same patients to be positive by culture year after year."

This statement was made like 14 months after the initial study was published. Also, that is exactly what was investigated in Phase IIb of the Blood Working Group: That same Judy (Mikovits) was free to chose her favorite 4 of the patients that were positive "year after year", then they mixed them up with a healthy control, and after that she couldn't tell these self chosen XMRV infected patients from the healthy, pedigreed negative patient (that was also self chosen and thus one of the patients that was "always consistently negative" in a non-blinded setting).

Admittably too little samples were used to draw definite conclusions from these results, but sure they were unlucky.

"We have found a patient to come back after 15 years and still be positive"
How does that argue against contamination in the slightest? Mix this patient up with 10 healthy and pedigreed negative controls (make sure the blood collection, storage and processing is exactly the same for all patients) and see if you can identify him from those samples.

Permalink to Comment

19. Myself on January 12, 2011 7:25 AM writes...

Yep, I gotta agree it looks messy. I still think the BREADTH of the evidence favors XMRV's existence, though. The antibody response, the cultures, the micrographs, the blinded studies, integration into human DNA, the PCRs, immunohistochemistry, and all the other tests used in the ME/CFS and prostate studies would all need explaining.

Interesting factoid about the sequence non-diversity finding. They didn't use WPI's samples, so if it's contamination, then it's their own lab's contamination. But yes, contamination could explain non-diversity. But then you'd have to explain why the entire lot was not contaminated (in the samples that were handled the same way). The labs could pull up their test records, and one could do a randomness analysis to see if there's patterns to the positive tests. There's so many other things I could add here, like WPI did run the CDC contamination tests, and a bunch of other things. Anyways, this is all so academic, even to a physicist.

In the end, they have to do what they are doing now .... more tests.

Good to see them moving to cultures, though.

Permalink to Comment

20. KAL on January 12, 2011 9:14 AM writes...

Actually it is a simple as this: patients don't make antibodies to lab contaminants.

Derek Lowe's blog comments seem particularly on target although he was not speaking of this particular wrangling.

"...this isn’t looking good at all. It’s pretty typical, though, of how things are out at the frontiers in this business. There are always more variables than you think, and more reasons to be wrong than you’ve counted.

A theory doesn’t hold up until everyone who wants to has had a chance to take some big piñata-shattering swings at it, with weapons of their choice.

So, to people outside of research: you’re not seeing evidence of bad faith, conspiracy, or stupidity here. You’re seeing exactly how science gets done. It isn’t pretty, but it gets results in the end."

Permalink to Comment

21. RRM on January 12, 2011 9:48 AM writes...

@Myself;

WPI did run the CDC contamination test, which only tests for mouse mitochondrial DNA. One thing that came out of the Retrovirology papers, is that another test (IAP) is a lot better than the CDC assay in detecting mouse contamination .

Another interesting thing in this context is the following: Mikovits and Ruscetti are now reportedly saying (through unofficial but reiliable channels) that this new, much more sensitive IAP test is not reliable, in the sense that it will (or at least can) also detect actual 'human XMRV' in samples. It will thus lead to false positive results for contamination in samples from patients that are actually infected with XMRV.

I find this last position to be intriguing. It seems to me that the only explanation for it would be that Mikovits and Ruscetti have checked some of their positive samples with the IAP assay and have gotten positive results. Of course, when you are convinced that you are right, you can then explain the finding away with believing the IAP assay did actually detect human XMRV instead of mouse DNA.

Another question that hasn't been raised by scientists: when were those WPI samples actually checked for mouse DNA? If they did check the actual samples before they were cultured (a procedure that was not described in the Science paper, but was later confirmed to have taken place in order to get the PCR results), it wouldn't mean much. You really have to check the cultured samples. This also goes for future testing, of course.

Finally, I suspect WPI have already cultured their samples for Phase IIb of the Blood Working Group. They wanted the best results, and it took them a whopping two months to report back on the few samples they were sent. Without much success, I might add.

@KAL:

It isn't as simple as "patients don't make antibodies to lab contaminants". You should only read the results of Blood Working Group Phase II (in which Ruscetti "detected" XMRV antibodies in a patient pedigreed negative for XMRV), as well as the negative UK study by Groom et al, which did find antibodies for XMRV in 14% of healthy controls and 0% of CFS patients.

I have no virology/serology background, but I would propose that when you contaminate your samples with "something" and subsequently culture your samples in the lab, simple evolutionary principles will dictate that antibodies to that "something" can/will form.

Permalink to Comment

22. Edugreat on January 12, 2011 10:28 AM writes...

RRM on January 12, 2011 9:48 AM writes...
I have no virology/serology background, but I would propose that when you contaminate your samples with "something" and subsequently culture your samples in the lab, simple evolutionary principles will dictate that antibodies to that "something" can/will form.

You are CORRECT to say you are no virologist, you are not an immunologist either..
Antibody formation involves more types of cells and the lymph glands which last I checked were not in the tube of blood drawn from my wife!

Antibody production can't happen in a test tube!

Permalink to Comment

23. cinderkeys on January 12, 2011 10:42 AM writes...

"Actually it is a simple as this: patients don't make antibodies to lab contaminants."

Also, if contamination accounted for the results in the positive studies' PCR test, then why wouldn't you see contamination causing false positives in both ME patients and healthy control subjects? Why, mysteriously, only the ME population?

Yes, science is messy. Yes, it's prudent not to leap to conclusions based on first results. But I find it odd that some members of the scientific community seem hellbent on dismissing the positive studies out of hand, using logic with holes large enough to drive a truck through. What's the motivation?

Permalink to Comment

24. RRM on January 12, 2011 11:02 AM writes...

I have learned that I should always listen to people who use caps and exclamation marks to convice people, so I stand corrected by your eloquent contribution. : )

Permalink to Comment

25. RRM on January 12, 2011 11:44 AM writes...

@cinderkeys:

Contamination actually resulting in more (false) positives in samples from patients than in samples from healty controls, is a phenomenon that has occured in the past. For instance, one study that was later found to be the result of contamination, was even "confirmed" two times by independent labs. Robin Weiss has written an excellent article about this.

The reasons for different results between patients and controls are not always clear, but it is assumed that it is the result of samples from patients handled more often than the samples from controls. Also, different blood collection protocols for the samples from patients and samples from controls could explain different results. It is interesting to note that in both positive studies, "banked" samples from patients were used.

The most important thing, however, is not how contamination can sometimes result in a higher rate of positives in patients' samples, but that it actually can happen.

My previous comment was not a reply to you, but a reply to Gerw...I mean Edugreat, by the way. : )

Permalink to Comment

26. urbantravels on January 12, 2011 1:03 PM writes...

Where I see evidence of bad faith is not in the published science itself, but in the misrepresentation of the conclusiveness of any one piece of evidence by the researchers themselves, and the subsequent echoing and amplication of those misrepresentations by the press and the Internet.

Such, I believe, was the case with the press release issued by the Wellcome Trust, which went so staggeringly far beyond the hypothesis presented (and so far unverified by other workers) in the Hue paper. To paraphrase Mark Twain, reports of the death of the XMRV/CFS hypothesis have been greatly exaggerated. It would help if the scientists themselves were not so eager to help write the premature obituaries.

Early science is messy and contradictory as hell and can get ugly; scientists, and even some informed laypeople, understand this, and can even see rigorous debate as a good and healthy sign. When information about the messy goings-on get filtered to the outside world via the press, however, either the mess is made to seem like mistakes, confusion and lack of progress (which we ought not to be wasting money on), or the mess is de-emphasized in favor of tidy “conclusions” that are often supported by privileging the claims of one side in a debate that may have multiple sides. (And if conclusions have been reached, we shouldn’t be wasting money on further investigation.)

We have seen how eagerly and uncritically the Wellcome Trust press release was carbon-copied into dozens of press outlets, blogs, Tweets, and spread around the world unchallenged. Why is this a concern, in the particular area of ME/CFS research? Because reporting premature conclusions in this matter serves to reinforce an old and very damaging narrative about CFS: that the cause is impossible to find; that repeated efforts to find the cause have all been blind alleys; that ME/CFS is a ‘vague and ill-defined’ illness, with the implication that an organic cause can never be found – and then we’re back in the “hysteria” wastebasket, with the century-old ideas of Charcot and Freud to weigh us down.

Public opinion affects support for research in very real terms; so does the official stance of the public health agencies. We have already seen several decades in which the NIH officially considered ME/CFS to be a form of depression or conversion disorder - based upon no particular hard evidence - and instructed the press to treat it accordingly. So there were few to protest when ME/CFS research was underfunded, or when funds intended for biological research were diverted elsewhere, or when grant applications for what little money was available were routinely turned down by ‘expert’ panels with no expertise in the disease. The NIH is showing encouraging signs of changing course in recent years, but we have yet to see the result in terms of hard dollars: is there any other disease as serious and disabling as ME/CFS that recieves so little NIH funding?

http://report.nih.gov/rcdc/categories/#bpopup

The recent article that appeared in Science magazine, though reasonably fair overall, ended on a rather sour note, which seems to me a pretty clear indication of how negative perceptions inside and outside the research community can start to lead to a chilling effect on research:

http://www.sciencemag.org/content/331/6013/17.full

“As the new [NIAID/Lipkin XMRV] study gets started, some wonder whether it’s worth the $1.3 million it will cost. Jonathan Stoye of the MRC National Institute for Medical Research in London concedes that the Towers study was “over-hyped.” But he says “it’s pointing people in a certain direction,” away from chasing an elusive link to XMRV. Still, he says, a larger study may be the only way to satisfy [CFS] patients.”

The journalist’s favorite gang of straw men, we all know, is “some.” Who are these “some” people who are already suggesting that $1.3 million is too handsome a sum to spend to get good answers on XMRV, in the face of these supposedly damning recent publications on contamination? Stoye seems to be saying that the only reason to proceed with the study, in his opinion, is to shut up the patients. This implication does a disservice to the NAID’s motives in ordering and funding the study. If XMRV truly does cause or contribute to human disease, then it’s not just the problem of a group of querulous patients who wants answers – it’s everybody’s problem, and NIH doing due diligence to look for those answers is the proper way to serve the public interest. To suggest otherwise - and in fact to suggest that research and debate should be ended when a question is far from settled - does, indeed, smack to me of bad faith.

Permalink to Comment

27. DzD on January 12, 2011 4:28 PM writes...

I would like to address several comments made by "RRM" in order to prevent the further spread of misinformation.

RRM: "Good write up. An additional, revealing piece of information that is not in the report by Science: In the first round of testing for the "project" (for the Blood Working Group), when two labs found XMRV, the blood was also collected by the scientists that had reported an asociation with XMRV. For the second round of testing, an independent phlebotomist visited the patients to collect new blood samples, and then all three labs found no XMRV."

I think you are mistaken here. First of all, blood was collected by an independent phlebotomist for BOTH rounds of Phase II of the Blood Working Group; as Graham Simmons said at the recent BPAC meeting:

(Simmons): "Moving on to the Phase IIa study, the WPI collected blood, using an independent phlebotomist, from four subjects...these were either processed immediately or put in 4 degrees for two or four days and then processed. Unblinded panels were distributed to the WPI and CDC and a blinded panel, including some XMRV-negatives, were sent to the DRP lab at NCI for testing."

It sounds as if he misspoke when he said "the WPI collected blood", because he later says panels were distributed TO the WPI. In any event, blood was not collected by the scientists themselves in the first round. My understanding is that the WPI contracted an independent phlebotomist to draw blood from their study participants and distribute it separately among the participating labs, including the WPI.

Second, in Phase IIb, the NCI reported positive results in serological assays. PCR was a murkier issue, but the BWG has decided to move ahead to Phase III in order to include a cohort size larger than 4 and to add serological testing for all samples as PCR seems unreliable so far. All this information and more is available from the FDA here: http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/BloodProductsAdvisoryCommittee/ucm239304.htm#p261

RRM: "It apparently is very reproducable in a non-blinded setting and it is at the limit of detection. I am sure I've read that before, and it usually is not a good sign."

Detection of XMRV or other MLV-related viral sequences has been reproduced in blinded settings in almost all cases. The WPI/NCI always blind their samples. Their recently reported study on UK patients was not only blinded but the samples were collected by an independent phlebotomist and sent directly to the NCI - and, if I'm not mistaken, another lab in the UK - with an even higher positivity rate in the patients than the Lombardi study but the same low rate in the controls. The Cornell study under Dr. Maureen Hanson is blinded, and so far their data has confirmed the findings of Lo et al. Three different researchers in Europe have found XMRV (including by different methods) in CFS patients as well.

RRM: "This is really, really strange. His/Lo's own study was not at all detecting MLV related sequences at the limits of detection. It used one of the least sensitive assays of all studies so far and they did PCR only. The Science authors also didn't initially report any problems to detect the virus."

This statement is incorrect. Please see Alter et al's response to comments in PNAS. Also, the WPI researchers DID report initial difficulties detecting the virus, as Mikovits et al have explained in at least two publications after the Lombardi paper's release. Furthermore, limit of detection is a term that is being applied to PCR in this case. It does not mean the virus is hard to find no matter what assay you use; it means that detection is much more dependent on particular reaction conditions.

RRM: "That same Judy (Mikovits) was free to chose her favorite 4 of the patients that were positive "year after year", then they mixed them up with a healthy control, and after that she couldn't tell these self chosen XMRV infected patients from the healthy, pedigreed negative patient (that was also self chosen and thus one of the patients that was "always consistently negative" in a non-blinded setting)."

You have misrepresented Alter's statement, even as you quoted it. He said positive by CULTURE, not by straight PCR. Mikovits was not doing culture in Phase II. Also, I believe by "year after year", Alter is referring to the fact that both fresh and banked samples of these patients taken over a course of many years have all proven positive by culture.

RRM: "'We have found a patient to come back after 15 years and still be positive'
How does that argue against contamination in the slightest? Mix this patient up with 10 healthy and pedigreed negative controls (make sure the blood collection, storage and processing is exactly the same for all patients) and see if you can identify him from those samples."

I will quote you Dr. Alter's answer to that question from the BPAC meeting.

"These were drawn in the clinic, sent directly to Dr. Lo, and analyzed immediately. If a contamination was coming from a reagent, these were samples drawn differently, the sequences had changed over time, and the controls were again negative. It just doesn't speak to me of a contamination."

By "the clinic" he is referring to Dr. Komaroff's clinic in Boston.

RRM: "WPI did run the CDC contamination test, which only tests for mouse mitochondrial DNA. One thing that came out of the Retrovirology papers, is that another test (IAP) is a lot better than the CDC assay in detecting mouse contamination ."

No, that did not come out of the Retrovirology papers. That mouse IAP assay is claimed to be more sensitive than the mtDNA assay by Coffin, but it has not been validated for this purpose. Again I refer you to the BPAC meeting - Lo and Coffin have an exchange on the issue; Lo had compared the mtDNA assay and the IAP assay on his own samples and (a) the IAP assay still found no contamination and (b) the mtDNA assay proved more sensitive.

RRM: "Another interesting thing in this context is the following: Mikovits and Ruscetti are now reportedly saying (through unofficial but reiliable channels) that this new, much more sensitive IAP test is not reliable, in the sense that it will (or at least can) also detect actual 'human XMRV' in samples. It will thus lead to false positive results for contamination in samples from patients that are actually infected with XMRV.
I find this last position to be intriguing. It seems to me that the only explanation for it would be that Mikovits and Ruscetti have checked some of their positive samples with the IAP assay and have gotten positive results. Of course, when you are convinced that you are right, you can then explain the finding away with believing the IAP assay did actually detect human XMRV instead of mouse DNA."

Idle speculation and implication of this sort is damaging and I think you should knock it off until you contact the researchers and ask them. Otherwise you are essentially smearing two very respected researchers. Furthermore, the 'explanation' they gave for their position in those emails is scientific data: recent work by Evans et al demonstrates that retroviruses can "package" mouse IAPs and carry these with them to any future host. To better understand this, I suggest you read Evans et al: "Mobilization of Endogenous Retroviruses in Mice after Infection with an Exogenous Retrovirus", Journal of Virology, March 2009.

RRM: "Finally, I suspect WPI have already cultured their samples for Phase IIb of the Blood Working Group. They wanted the best results, and it took them a whopping two months to report back on the few samples they were sent. Without much success, I might add."

If you would use some of your energy to contact the WPI instead of spamming an internet blog, you wouldn't have to "suspect" anything - you might actually know FACTS. And again, Dr. Simmons confirmed that the WPI did not do culture in Phase II. For you to publicly state otherwise is an accusation of scientific fraud in a government task force study. Do you realize how serious that is, and how liable you are making yourself for damages?

RRM: "I have no virology/serology background, but I would propose that when you contaminate your samples with "something" and subsequently culture your samples in the lab, simple evolutionary principles will dictate that antibodies to that "something" can/will form."

Well, I actually do have a 'serology background', and I must inform you that what you "propose" is complete nonsense. That is not how antibody production works at all - it CANNOT happen in the test tube, but rather requires a complex interaction between specific immune cells in particular parts of the body. Why would you enter into a serious scientific discussion of an issue of great importance to millions of people and simply make stuff up? That's shameful and disturbing, and I suggest you consider the ethics of what you are doing.

About the issue of contamination being higher in patient samples than controls - you cited Weiss's paper, which I am very familiar with. You also cite one example as a general principle - one exceptional case as a rule. That is not scientific. Weiss and others have made the point that they cannot explain the phenomenon of that case or any like it, and they only speculate about the handling issue. You are also incorrect that the Lo study used only banked samples, as the more recent samples they tested were freshly drawn after the paper was initially submitted to PNAS. Further, the WPI UK study and others that have been presented at conferences used only freshly drawn samples that were fully blinded, which eliminates both your concerns.

Lastly...I could be mistaken, but I was under the impression that you a journalist of sorts (?). If that is the case, I suggest you contact Dr. Alter directly for information rather than posting opinion on his statements. Otherwise, by posting in this manner on various blogs, you would be seriously damaging your credibility as an objective journalist. Even if that is not the case, I still recommend you do your homework and perhaps learn more about immunology before analyzing any scientific data or publicly questioning the integrity of any scientist. You appear to be unqualified to do either.

Permalink to Comment

28. Edugreat on January 12, 2011 8:17 PM writes...

@RRM:
I would like to clarify that my wife tested negative for XMRV using the VIP Dx lab in May '10 where it took them 30 - 45 day to culture the blood and do the multiple tests etc. on it - she then tested positive for the antibodies (using the same blood draw) in December '10. WPI and the lab asked if we would be interested in having the blood retested for the antibodies since they had found that the retrovirus seemed to enter the cells rapidly upon infection and might not show up in the original test -so once they had the serology test ready they were retesting the blood to see if antibodies were present and thus infection had taken place.
Here blood was drawn in the LA area by a lab of our choosing and sent overnight FedEx - not iced to the VIP Dx lab.
If her results are because of contamination please explain how?
And yes she has CFS - but while we hope XMRV or some other MLV is the cause so that treatment options can begin to be established, we are open to other or co-causal factors. The main thing out of all this is that her Dr.s are now more ready to acknowlede CFS as a biological disease with a biological cause, most likely viral.

--- how was that - no CAPITALS or !!!! :-)

Permalink to Comment

29. RRM on January 13, 2011 3:47 PM writes...

@Dzd:

Thank you for your substantial and (mostly) eloquent reply, and that is without irony.

First, I am not a journalist at all. If you wanted to actually know my background in this you could have asked for it, instead of "suspecting" things yourself.

Second, I got my blood collection information from the slides posted on the CFDIS site , which literaly state that:

"WPI collected blood from four subjects (mostly with CFS) previously identified as XMRV positive in the Lombardi et al. study (by PCR, serology and/or culture). Specimens separated into tubes and were processed immediately, or left at 4oC for 2 or 4 days. Each specimen was processed into replicate peripheral blood mononuclear cells, whole blood, and plasma samples that were frozen for panel preparation."

So it seems Graham Simmons also "misspoke" when he made this slide (slide 24). I will add that I never meant to imply that the actual scientists themselves visited those patients.

"Second, in Phase IIb, the NCI reported positive results in serological assays"

Yes, they reported 5/8 XMRV+ pedigreed patients to be positive, and 1/2 negative samples. I am not saying these findings are false, but it seem to be about the same score a monkey would get. Actually, the WPI's and NCI's combined NAT/serology results for Phase IIb were exactly that (see slide 36): cumulatively, they scored 8/24 for pedigreed positives and 2/6 for pedigreed negatives. Color me not impressed.

"Detection of XMRV or other MLV-related viral sequences has been reproduced in blinded settings in almost all cases."

Where is the published data to support these assertions? Why doesn't WPI or any other lab that can detect this thing get their harshest critics to collect the samples? If I were Judy Mikovits, instead of posting "rebuttals" on WPI's website, I'd call Myra McClure (or Wessely, Reeves, Vd Meer, whoever) to supply me with some samples, and convince them by solidly detecting the CFS "needles" from the healthy "hay".

"This statement is incorrect. Please see Alter et al's response to comments in PNAS."

I made several statements, so I wouldn't know what to make of this. Regardless, I think the general sentiment I expressed about Alter's "limit of detection" is true: if it were THAT hard to find as now asserted by those who are able to find it, the initial findings wouldn't had been what they were.

"You have misrepresented Alter's statement, even as you quoted it. He said positive by CULTURE, not by straight PCR. Mikovits was not doing culture in Phase II."

Let's see. From slide 25 of the presentation I've alreay quoted from: Mikovits chose four patients that collectively tested positive for PCR 16 out of 17 times. They were "only" culture positive 20 out of 23 times. So I would say that the statement that "Judy (Mikovits) was free to chose her favorite 4 of the patients that were positive "year after year", then they mixed them up with a healthy control, and after that she couldn't tell these self chosen XMRV infected patients from the healthy, pedigreed negative patient" is pretty accurate.

"Alter is referring to the fact that both fresh and banked samples of these patients taken over a course of many years have all proven positive by culture."

I readily admit that am not natively English (I have in fact never even visited an English speaking country), but I would say that your interpretation is either incorrect, or of course Alter misspoke (in which case it still remains a strange statement). Year after year means "for many years, one year after another" and not "two moments that are many years apart".

"I will quote you Dr. Alter's answer to that question from the BPAC meeting."

I will also quote you Dr Alter from the FDA Q&A: "We did not specifically blind and mix the two sample groups (CFS and blood donors)"

I will thus repeat: MIX this patient up with 10 healthy and pedigreed negative controls (make sure the blood collection, storage and processing is exactly the same for all patients) and see if you can identify him from those samples.

"No, that did not come out of the Retrovirology papers.[...]Again I refer you to the BPAC meeting"

Still, some of the Retrovirology IAP data came from a different study than the one from Coffin. The other study stated that:

"We found the mtDNA PCR 100-fold less sensitive than that for IAP."

...and...

"These results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR".

You seem to be cherry picking unverified, unreviewed and perhaps outdated evidence to support your argument.

"recent work by Evans et al demonstrates that retroviruses can "package" mouse IAPs and carry these with them to any future host."

Then I regret saying this. However, I will state that the reliable and knowledgable source on this was saying that there was no published evidence for this possibility.

And still, it doesn't makes real sense. If the above is true, then we actually have the best proof for XMRV in or hands! Mikovits could (again) use this brilliant IAP assy to actually pick the sick "needles" from the healthy "hay" (all provided to her by Simon Wessely, using the exact same protocols for patients and controls, blood collected in the same room). She could have this mystery solved in mere weeks!

"For you to publicly state otherwise is an accusation of scientific fraud in a government task force study. Do you realize how serious that is, and how liable you are making yourself for damages?"

You may have a background in serology, but I can assure you from my background that I have not myself liable for damages in any way.

Instead of me, you have just (thus rather ironically) implicitly accused some people (the original Science authors) of committing scientific fraud by stating that it is scientific fraud to actually culture samples while stating that you've just done PCR. ;-)

I'll just restate that it is very hard for me to believe that WPI took ~60 days to (partially) report back on the results of PCR. In contrast, it took them less than a month to report back on their PCR results for Phase I, which I believe consisted of more samples.

"Why would you enter into a serious scientific discussion of an issue of great importance to millions of people and simply make stuff up? That's shameful and disturbing, and I suggest you consider the ethics of what you are doing."

Wow. Here you do disqualify yourself. I did not make stuff up. I first stated that I was not an expert in the field by any means and then made a suggestion on simple logical grounds (that is, for a laymen). Everybody with any basic knowledge of the English language could understand it was merely a suggestion by someone without specific knowledge in the matter by reading the quote in the proper context.

Because the original authors of the Science paper were very unclear about culturing in their study, I just felt it might fly under the radar if it were an issue with culturing.

"You also cite one example as a general principle - one exceptional case as a rule. That is not scientific."

You seem to be losing yourself a bit in the end (see also the previous quote). Again, I referered to the full paper by Weiss and then gave an example from the paper, which I introduced by the words "for instance". How is that "not scientific"?

Also, if someone proposes or implies that an event is "impossible", it would only take one example to prove that statement to be incorrect. I suggest you (re-)read the post I responded to and rationally reconsider your statement.

For your information, both Judy "they skewed their experimental design in order to not find XMRV in the blood" Mikovits and Frank "I do not know how they think they get away with this ethically" Ruscetti have not only questioned but downright attacked other scientists that didn't get the same results as them.

@Edugreat:

It could have THE REAGENTS!!!


Permalink to Comment

30. Dv on January 13, 2011 8:48 PM writes...

RRM, you are jumping the gun..again. And as you are not a scientist it is easy to see why. Your monkey comment is pathetic, and cannot be regarded as an argument.

Mikovits on serology results "There was complete concordance between Frank Ruscetti's serology results and our work with those patient samples. We don't do direct PCR. We were asked to do that for the purpose of this study."
'December 14, 2010: Blood Products Advisory Committee Meeting Transcript'

The WPI has already offered to take samples from McClure, and vice versa. If you don't know this, why are you commenting?

Lombardi et al. was not unclear on culturing, you are cherry picking.

As a new positive study will be out soon, your whole raison d'etre is waste of everyones time.

A couple of days ago you thought it was the reagents that were tested in Sato et al. not a PCR testing kit.

You do not provide sources for much, and you come off as a fool. And dull

Permalink to Comment

31. Richard Jefferys on January 14, 2011 1:03 AM writes...

"There was complete concordance between Frank Ruscetti's serology results and our work with those patient samples"

According to the webinar, Frank Ruscetti's serology got both positive and negative results on the pedigreed negative control, and got positive and negative results on day 0 and day 2 plasma samples from a putatively infected individual from the original Science paper. Is Mikovits saying that WPI got the same? If so, what would that mean? That the assay is reliably uninterpretable?

Do you have any insights as to if and when data on XMRV-specific CD4 and CD8 T cell responses may be forthcoming?

Permalink to Comment

32. Anonymous on January 14, 2011 12:19 PM writes...

-->"Your monkey comment is pathetic, and cannot be regarded as an argument."

It should really not be regarded as an argument, as it is a factual statement. For your information, Mikovits herself did even worse than an actual monkey would do on average. If you check slide 36 again, you'd see that she detected XMRV in 3 out of 16 (19%) well pedigreed XMRV+ patients, and in 1 out of 4 (25%) well pedigreed XMRV- negative patients.

-->"Mikovits on serology results: "There was complete concordance between Frank Ruscetti's serology results and our work with those patient samples.""

Mikovits is not referring to any blinded test that was done in the setting of the Blood Working Group. It actually looks like she is referring to the pedigreeing of those samples before the actual testing, and it would be a VERY STUPID statement if that suspicion were true. I'll explain why:

From all the samples you test, of course you will only pick the samples that are in concordance with each other for blinded retesting, and of course, BY MERE CHANCE, some of the samples will be in concordance with each other if you test enough samples.

When after that, your results don't hold up in a blinded setting, it implies you have a HUGE problem with your hypothesis (or just very, very bad luck).

I cannot believe that you think Mikovits' statement somehow supports your argument. If anything, if the above is correct, it should show you that Judy is foolishly arguing in circles (I am actually being kind here).

-->"Mikovits: "We don't do direct PCR. We were asked to do that for the purpose of this study.""

Mikovits (and/or Ruscetti) tested those 4 patients beforehand, using JUST PCR, 17 times and the samples tested positive, using JUST PCR, 16 times.

After that, she couldn't identify the positive samples from the negative samples in a blinded setting.

Yet this does not worry you at all?

-->"The WPI has already offered to take samples from McClure, and vice versa. If you don't know this, why are you commenting?"

The WPI didn't offer to do what I proposed. They once offered to exchange samples and reagents. I'll repeat: if Mikovits/Ruscetti/Lo were right, they could have solved this mystery using my simple and cheap methodology months ago. Also, there are others besides McClure (Vd Meer, Wessely, Reeves, should I go on?) who could have helped you convince the world by just collecting samples for you.

-->"Lombardi et al. was not unclear on culturing, you are cherry picking."

Lombardi et al: "we isolated nucleic acids from PBMCs and assayed the samples for XMRV gag sequences by nested polymerase chain reaction (PCR) (5, 6). Of the 101 CFS samples analyzed, 68 (67%) contained XMRV gag sequence"

Please cherry pick the lines from the paper in which they stated (or implied) that they had cultured their samples to reach their results.

-->"As a new positive study will be out soon, your whole raison d'etre is waste of everyones time."

Ah, the future studies will prove everybody wrong argument. Even if 'a new positive study will be out soon', we will have to wait and see how those results hold up. Whether you think the Lo/Alter paper confirms the Lombardi et al findings or not, you'd have to admit that it wasn't exactly the convincing solution that many people were expecting. Which should show you that, in science, you take your position based on weighing the *available* evidence. But you knew that, right?

-->"A couple of days ago you thought it was the reagents that were tested in Sato et al. not a PCR testing kit."

You repeat the errors of others. Instead of arguing, I will just post a comment on the Sato study from an interview with Eric Klein form the Cleveland Clinic:

"Maupin: One criticism was that contamination affected the PCR REAGENTS employed in testing kits.

Klein: Yes. We noticed that and we did some experiments with that REAGENT a few years ago, so we knew that was a problem and we stopped using it because of it. So, that is a cautionary tale: don't use that REAGENT. That is the only thing you can draw from that paper."

So I guess it is official that you think that Eric Klein is a bad scientist for making such stupid errors?

-->"You do not provide sources for much, and you come off as a fool. And dull"

Thank you for your opinion, because I really care how you think about me. As for sources: if any of sources is too difficult for you to find, I would gladly direct you to it.

Permalink to Comment

33. RRM on January 14, 2011 12:20 PM writes...

I am sorry, the above is by me (RRM).

Permalink to Comment

34. cinderkeys on January 14, 2011 12:38 PM writes...

"The reasons for different results between patients and controls are not always clear, but it is assumed that it is the result of samples from patients handled more often than the samples from controls."

That's one hypothesis. Another hypothesis is that the independent variable caused different results between patients and controls.

Remember, the four recent studies only showed that contamination occurred in their own experiments, suggesting that contamination can occur elsewhere if researchers aren't extremely careful. This is not new information, and assuming contamination due to unequal handling in the actual positive studies seems a bit premature.

Permalink to Comment

35. JC on January 15, 2011 5:34 AM writes...

In post 18, RRM says:
"Are there many real-life examples where whole groups of scientists were biased agianst a new finding that turned out to be correct?"

Hi RRM. You are kidding me, aren't you? No? Ok - Galileo, Copernicus, Darwin's evolutionary theories still cause a stink in some places, Warren and Marshall and the causation and cure of stomach ulcers.... read about that, it has rather close parallels with ME/CFS and XMRV (though we haven't got to the final accepted truth in this field - yet).

"1975
Steer and Colin-Jones publish their results regarding H. pylori and its relation to PUD*. They decide that it was Pseudomonas, a contaminant, and not related to PUD."

"2005
Warren and Marshall are awarded the Nobel Prize in Physiology or Medicine for their work on H. pylori and PUD."

*PUD = peptic ulcer disease
http://en.wikipedia.org/wiki/Timeline_of_peptic_ulcer_disease_and_Helicobacter_pylori

Please, think about it, what you do matters to the world and changes it. Don't be one of the forces that mean we have to wait for help for another three decades.

Permalink to Comment

36. Dv on January 15, 2011 12:38 PM writes...

And how would you know that a sample is negative at this stage in the game? It's a bit early for absolutes.

Permalink to Comment

37. Anonymous on January 15, 2011 12:41 PM writes...

"Do you have any insights as to if and when data on XMRV-specific CD4 and CD8 T cell responses may be forthcoming?"

Should be soon.

Permalink to Comment

38. Dv on January 15, 2011 1:19 PM writes...

RRM you don't really have anything to say do you, and not being a scientist why would you.

In the early days of HIV 30% of positives were missed. As MRV testing improves, so will the numbers of those detected. You cannot therefore assume that the negative sample to be actually negative. Only further research will.

The fact that Ruscetti's and Mikovits serology results match in Phase IIb is not being ignored by the Blood XMRV working group. It means something. Which samples the WPI chose to provide to the study at that time has no bearing on the results. Mikovits can't fix Ruscetti's blinded study results now can she.

The WPI do not use direct PCR for detecting positives. They were asked for the BWG study. What do you not get? They haven't developed that test. It is the least sensitive method available, and no one should be relying on it. You do realise that viral load will also alter over time don't you?

The WPI have always offered to test samples from others and have done so. What are you complaining about? It is those very people you list who are not helping to solve the problem and are stalling. What are they afraid of?

CULTURING:
I guess you only read the start of the paper, that was your first mistake. Here are two bits. There are more.

"We next investigated whether the viral pro- teins detected in PBMCs from CFS patients rep- resent infectious XMRV. Activated lymphocytes (6) were cocultured with LNCaP, a prostate can- cer cell line with defects in both the JAK-STAT and RNase L pathways (10, 11) that was previ-
ously shown to be permissive for XMRV infec- tion (12). After coculture with activated PBMCs from CFS patients, LNCaP cells expressed XMRV Env and multiple XMRV Gag proteins when analyzed by Western blot (Fig. 3A) and IFC (fig. S5A). Transmission electron microscopy (EM) of the infected LNCaP cells (Fig. 3B), as well as virus preparations from these cells (Fig. 3C), revealed 90- to 100-nm-diameter budding particles con- sistent with a gamma (type C) retrovirus (13)."

"Viral transmission. Frozen cell-free plasma and 0.22 μm filtered cell free supernatants from PBMC and T cell cultures were diluted 1:1 with tissue culture media and 600 μL aliquots were added to a six-well culture plate with the LNCaP cell line (50% confluent) or a million primary activated CD4+ T cells isolated from healthy donors. The plates were centrifuged for 5 min at 1500 RPM, rotated 180o and centrifuged again for 5 min. The entire cycle was repeated once and cells were then diluted in their growth media. For cell-cell transmission, 1 x 106 T cells or PBMC without any IL-2 in the growth media were added to a six-well culture plate with the LNCaP cell line (50% confluent) in 1 mL of media for 3 h. After 1 hr, T cells in suspension were removed and the LNCaP cells were grown for several passages in the absence of IL-2 which caused any remaining T cells to die. At the times after transmission indicated, protein analysis was done by western blot and flow cytometry."

More positive studies are on the way, just like Lo et al. and Lombardi et al. Still means the same thing as when I wrote it, but you knew that right?

But you thought that Lombardi et al had used that PCR kit didn't you. Didn't know the difference did you. Sato et al. didn't even identify the virus they had detected did they, therefore that paper is of no importance.

Couldn't be bothered is my assessment of your analysis

Permalink to Comment

39. Anonymous on January 15, 2011 1:25 PM writes...

Isolation, separation and culture of primary cells. Leukopaks of peripheral blood from healthy donors were collected according to a NIH approved IRB #99- CC-0168 protocol. Patients’ peripheral blood and plasma samples were from frozen banked samples obtained under NIH exempt status. Mononuclear leukocytes from both normal and patients’ cells were isolated by Ficoll-Hypaque gradient centrifugation. The light density fraction (buffy coat) was collected and washed twice with PBS. PBMC were activated by 1 μg/mL PHA (Abbott Diagnostics, Abbott Park, IL) and after 72 hours the cells were cultured with 20 units/mL of IL-2 (Zeptometrix, Buffalo, NY) and subcultured every 3-5 days. For isolation of CD4+T cells, CD8, CD11b, CD14, CD19, CD33 and CD56 positive cells were removed using magnetic activated cell sorting (MACs) methods according to the manufacturer’s instructions (Miltenyi Biotec, Inc., Auburn, CA). After isolation, the CD3+, CD4+ T cells (>95% pure) were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate and antibiotics. CD4+ T cells were activated by culturing with 20 units/mL of IL-2 and 1 μg/mL PHA.
In vitro expansion of primary B-cells. NIH 3T3 cells transduced with a retroviral vector expressing CD40L (gift of Eugene Barsov, NCI-Frederick) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% calf serum (CS) (Lonza, Basel, Switzerland) and 1% penicillin, streptomycin and L-glutamine (Invitrogen, Carlsbad, CA) at 37°C with 5% CO2. To stimulate B cell expansion, ~3.5 x 106 NIH3T3-CD40L cells were trypsinized (0.25% trypsin with EDTA )(Invitrogen, Carlsbad, CA), resuspended in 3 mL medium and irradiated with an absorbed radiation dose (rad) of 9600 using a Cesium137 irradiator. Cells plus 7 mL medium were added to a T25 cell culture flask (Corning, Corning, NY) and allowed to adhere (2-3 h) to the flask surface (optimal density ~50%).
CD19+ B cells were isolated from PBMC using immunomagnetic bead technology (Miltenyi Biotec, Auburn, CA). CD19+ cells were separated from 108 freshly isolated PBMC by positive selection according to the manufacturer’s protocol. After magnetic separation, CD19+ B cells (>95% pure) were added to
4
an irradiated NIH3T3-CD40L monolayer and incubated at 37 °C with 5% CO2. Cultures were monitored for B cell proliferation and split 1:5 every 72-96 hr onto freshly irradiated NIH 3T3-CD40L monolayer. CD19+ primary B cells were cultured and expanded in primary B cell expansion media: Iscove’s Modified Dulbecco’s Medium (IMDM) (Invitrogen, Carlsbad, CA) + 10% FCS (Atlanta Biologicals, Lawrenceville, GA), 1% penicillin, streptomycin and L-glutamine (Invitrogen, Carlsbad, CA), 40 ng/mL interleukin 4 (IL-4) (PeproTech, Inc., Rocky Hill, NJ), 50 μg/mL holo-transferrin (Sigma, St. Louis, MO) and 5 μg/mL insulin (Invitrogen, Carlsbad, CA).
Cell culture and reagents. Raji, SupT1 and LNCaP were obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in RPMI-1640 supplemented with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 ng/mL), and FCS (10%) and subcultured 1:5 every 4-5 days. HCD-57 cells are a mouse erythroleukemia cell line that expresses both ecotropic and polytropic MLVs; HCD-57/SFFV are HCD-57 cells infected with SFFV. BaF3-ER cells are a murine pro B cell line engineered to express the erythropoietin receptor. BaF3ER-SFFV Env cells were derived and maintained as described (S6).
Flow cytometry for viral proteins. Adherent cells were incubated in trypsin for 10 minutes at 37oC. After additional washes, adherent and suspension cells were incubated for 15 min at RT in 1 mL of paraformaldehyde (4% w/v in PBS), washed in permeabilization wash buffer (0.5% saponin 0.1%, sodium azide, 2% human AB sera in PBS) (PWB), and resuspended in 300 μL of permeabilization buffer (PBS with 2.5% saponin) (PB). After incubating at 22oC for 20 min, 5 mL of human AB sera and either rat anti-MLV p30 mAb, rat anti-SFFV Env mAb, goat anti-Rauscher MLV gp70 Env, p30 Gag, or p10 Gag, or the appropriate isotype control (anti-rat IgG, rat myeloma supernatant, or preimmune goat serum) were added, and the cells incubated at 4oC for an additional 30 min. Cells were then washed in PWB, resuspended in 100 μL of PB with 3 μL (0.6 μg) of FITC- conjugated goat anti-rat IgG or rabbit anti-goat antibody (BD PharMingen, San Jose, CA) and incubated for 20 min at 4oC. The efficiency of permeabilization was determined using a FITC-conjugated anti-actin antibody. Cells were then washed twice in PB, resuspended in 500 μL of sheath fluid (BD PharMingen, San Jose, CA) to prevent clumping and analyzed by flow cytometry. For experiments in which purified cell populations were examined, cells were stained with an anti- CD3 or anti-CD19 antibody prior to permeabilization, and analyzed by gating on the CD3+ or CD19+ subsets.

Permalink to Comment

40. Richard Jefferys on January 15, 2011 1:37 PM writes...

"And how would you know that a sample is negative at this stage in the game? It's a bit early for absolutes."

Is this what the people paying money for these antibody tests are being told? There were different results from day 0 and day 2 samples from the same person, in two instances.

That test should not be for sale "at this stage in the game."

Permalink to Comment

41. Anonymous on January 15, 2011 3:57 PM writes...

@Richard Jeffreys

About the "There was complete concordance between Frank Ruscetti's serology results and our work with those patient samples" samples by Mikovits:

The results Judy Mikovits referred to were not part of the Blood Working Group results. It actually seems rather hilarious:

Judy Mikovits could choose the four patients from her Science patient population of 101 CFS patients (67 initially positive for XMRV, later asserted to be 99 or 100) in which XMRV was easiest to detect. Thus she chose those patients in which the PCR, culture and serology results were in 'complete concordance' with each other. You can see how well the results were in concordance with each other before blinded testing here: http://www.cfids.org/webinar/slides-121710.pdf.

Now, the blinded testing fails (see slide 36). Mikovits actually does worse than actual chance would predict. According to her results, being XMRV positive is correlated to being XMRV negative!

How does she defends these findings? By telling us that "there was complete concordance between Frank Ruscetti's serology results and our work with those patient samples".

This is laughable. If her argument is 'we chose these samples for blinded testing because the results of these samples were in concordance with each other and, despite the fact that we couldn't tell these samples apart from pedigreed negatives in a blinded setting, they must be correct, because they were in concordance with each other', it would be about the worst argument I have ever heard.

@Dv

"I guess you only read the start of the paper, that was your first mistake. Here are two bits. There are more."

No, it wasn't and no, there aren't. The part you quote from has nothing to do with culturing samples to find XMRV. The often used English word "next" should have really put you on the right track there.

Never in the paper it is asserted or implied that the 67% number was established with culturing.

Also, I have never said that Lombardi et al used the very same PCR kit. If you read back, you'll see that I stated that they used the same BRAND. Sato's contaminated kit and the reagents therein were from Invitrogen, just as the kits (and reagents therein) used by both Lombardi et al and Lo et al.

The rest of your post is mostly unintelligible and doesn't address the points I have made earlier in reply to your earlier comments.

Permalink to Comment

42. RRM on January 15, 2011 3:59 PM writes...

"You can see how well the results were in concordance with each other before blinded testing here: http://www.cfids.org/webinar/slides-121710.pdf."

I forgot to add: see slide 25

Permalink to Comment

43. ERV on January 16, 2011 12:46 PM writes...

The deal with the 'anti-XMRV antibodies' is something we have been dealing with in HIV-testing for decades, and has been explored in previous papers.

Everyone makes antibodies to everything. That is how your immune system works. If you never 'see' an invader, you can never make a *potent* immune response to said invader. Thus, even a virgin who has never been exposed to any needle, ever could walk into a clinic one day, take a fast-HIV test (detects anti-antibodies in saliva), and come up 'HIV positive'.

That is why antibody tests are not the be-all-end-all-final-diagnosis tool for HIV-1 infection.

Nor is 1950-esque 'viral culture'.

Real-Time quantitative PCR is.

Because this is 2011.

Furthermore, that virgin who tests 'positive' one day might next negative with the very same test a week later. Maybe they were just getting over a cold. Maybe their allergies were acting up. An immune disregulation could knock things off balance. Or maybe it was just a transient occurrence.

In any case, it seems clear that the patients chosen for the initial paper had immune system abnormalities, while other papers chose people with a CFS diagnosis. Immune disregulation could lead to 'anti-XMRV-antibodies', or 'anti-HIV-antibodies', or 'anti-anything-we-are-looking-for-antibodies' that are not real. They are not really in response to infection, but a sign of immune annoyances or simply chance.

The anti-XMRV antibodies isolated *WERE NOT REAL*.

We know this, because another lab *also* found 'anti-XMRV-antibodies' (more in their controls than their CFS patients). They then investigated whether those antibodies were able to neutralize XMRV.

They were not.

Which means they were antibody-noise, not a real antibody response in response to a real infection.

Now, I do not expect any person outside HIV-world/immunology to know this. Certainly not CFS patients, certainly not Average Joes/Janes. Makes one wonder why Judy Mikovits, who is not an Average Jane, does not know this information. Or at least pretends not to know this information when addressing her employers and CFS patients.

Permalink to Comment

44. RRM on January 17, 2011 9:02 AM writes...

@JC

Sorry, I hadn't read your comment.

JC--> "Hi RRM. You are kidding me, aren't you? No? Ok - Galileo, Copernicus, Darwin's evolutionary theories still cause a stink in some places."

Galileo, Copernicus and Darwin were not opposed by a majority group of mainstream scientists at all. They were mostly opposed by the religious establishment of the time.

JC--> "Warren and Marshall and the causation and cure of stomach ulcers.... read about that.."

Are YOU kidding me? If you read back my post you quote from, you will see that I included that exact same example (as it is the only one I could think of). And even then, I wouldn't call it a very good example in this context, because other scientists then didn't really "refuted" those findings with a failure to duplicate the original results - the findings were "just" largely ignored by many mainstream scientists.


JC--> "it has rather close parallels with ME/CFS and XMRV (though we haven't got to the final accepted truth in this field - yet)."

You might want to read the following, to prevent future logical fallacies from happening:
http://oracknows.blogspot.com/2005/03/galileo-gambit.html
Or if you don't want to read it, this Carl Sagan quote explains it in the easiest way possible:

"They laughed at Copernicus. They laughed at the Wright Brothers. Yes, well, they also laughed at the Marx Brothers. Being laughed at does not mean you are right."

Permalink to Comment

45. Edugreat on January 17, 2011 9:28 PM writes...

So let me get this straight..
If some one tests negative for XMRV the test is valid, if they test positive there was contamination and the result should be thrown out.

if they have antibodies it is "noise" bad regents some other way false positive, but if they show no antibodies then the results are valid..

and the labs who find XMRV / antibodies are biased and would find it anywhere they look, but the labs who don't (can't) find it are the only true scientists and show no bias what-so-ever

That PCRs are the only valid way of finding viruses unless they find XMRV, then they are contaminated.

Wow - that is so clear now - I'll go tell my wife it really is all in her head...

Permalink to Comment

46. ERV on January 17, 2011 9:52 PM writes...

There are details to these tests that you dont understand because its not my job or responsibility to make sure you understand these tests. Its the XMRV+ labs jobs (whos research is paid for by your tax dollars, btw).

Im nice, so I dont mind taking a moment to explain it to you, but its hard for me to get the momentum/initiative when instead of saying "I dont get it", you actively chose to be a jerk.

For the lurkers:
The antibody 'test' the WPI used is a Western Blot. You take viruses, blow them up, and you look for antibodies that 'see' the guts. If you look at figure 4 in the Science paper, there are lots and lots and lots of bands in their gels that were supposed to be for one thing-- XMRV env or XMRV gag. But... there were lots and lots and lots of bands? Because of non-specific crap... so how do you know what is crap and what is real?

When Abbot framed an XMRV Western test off the HIV-1 Western test, they did not count someone as 'positive' until their sera had antibodies to three different proteins (it ended up being 3 people out of ~3,000-ish?).

What is better, if you have a gun to your head and you MUST look for antibodies and nothing else, is to see whether those antibodies actually recognize and neutralize real virus. Not viral guts. Real virus. Every HIV-1 patient on Planet Earth makes neutralizing HIV-1 antibodies... its just that its not enough, and they are too late to prevent disease. Its a standard test I do all the time.

Anyway, when scientists mixed their 'XMRV positive' sera samples with real, live, XMRV (not guts), it had no effect on the virus. AAAAAAALL those 'anti-XMRV-env' antibodies, and not one virus was neutralized? Defies logic.

Unless the antibodies are, in fact, non-specific.

Permalink to Comment

47. Edugreat on January 18, 2011 3:07 PM writes...

to ERV:
my cynical statements in my above post was more to point out the view from the patient which I feel is often overlooked with all the bickering and tit for tat in the above posts. You state that "Its the XMRV+ labs jobs (whos research is paid for by your tax dollars, btw)." well what those of us who are dealing with the disease on a personal level know is that "tax dollars" have been few and far between over the last 30 years with CFS research buried at the CDC. It was only when private money built the WPI and they began extensive multi-pronged research into CFS (causes and treatments) that the Science XMRV paper was published. Now CFS is in the news, blood supplies are protected, and research is being done at multiple labs worldwide. For what it is worth - I could take or leave an XMRV connection at this time - finally research is being done and CFS is in the news.
What has been most upsetting is to see four papers who come out and in bold headlines say that "XMRV is NOT the cause of CFS" - and discredit all work done over the last few years as the result of "poor" science methodology, but who fail to give any soild evidence that contamination actually occurred, just that it could have.
Nobody at WPI or any other lab has stated the XMRV DOES CAUSE CFS - only that it appears related somehow and bears more research done.
It is interesting that the WPI scientists have had their reputations slandered in many of the above posts, but a deaf ear is turned to questions about the links of the Wellcome trust which funded some of the four papers and yet is also in process of doing a study on the effectiveness of psyco - behavioral therapy to treat CFS since it according to them not a manifestation of any type of biologically (viral...) mediated disease.
for more on this Google "wellcome PACE Trial" and see the "quality" of their science.
I do have a background in science and understand the limitations of different methodologies being used - but as a husband to a CFS sufferer I have come to understand many in the science community have lost the most important tools of insight and intuition, which have often been the keys to unique discoveries.

Permalink to Comment

48. Anonymous on January 18, 2011 4:43 PM writes...

"Now, I do not expect any person outside HIV-world/immunology to know this. Certainly not CFS patients, certainly not Average Joes/Janes."

ERV why would researchers and doctors not get CFS?

Permalink to Comment

49. Anonymous on January 19, 2011 6:17 AM writes...

Anonymous: "Never in the paper it is asserted or implied that the 67% number was established with culturing." Correct, as it was the result of using multiple technologies:

a) PCR on nucleic acids from un-stimulated and stimulated white blood cells;
b) XMRV protein expression from stimulated white blood cells;
c) Virus isolation on the LNCaP cell line; and
d) A specific antibody response to XMRV.

RRM in response to Hohn et al. did say that Lombardi et al. used the same kit. As you don't reveal your name, I must assume you are RRM, and is must be another RRM who is less bright. BRAND is obviously not important if the kit was tested for contamination before use by Lombardi or Lo et al. Sato et al. never did retest their CFS patients after they discovered that particular kit to be contaminated with some sort of virus. Guess they just gave up.

ERV you are quiet right. Lombardi et al. was studying those with ME, a neurological disease. The negative papers are looking at general fatigue or even chronic fatigue, AKA CFS.

The other lab you refer to, or Groom et al. admitted "that their neutralization assay did not detect bacterially expressed XMRV gag and that positive control sera was needed to validate their assay. The WPI’s monoclonal antibodies specifically and sensitively completed the immune response demonstrating the assays sensitivity and specificity for XMRV envelope."

As Lombardi et al. was blinded, and accepted by Science. The bands of which you are so keen to highlight are not really important now are they.

Permalink to Comment

50. Camaro on January 19, 2011 9:18 AM writes...

The multiple techniques in the original Lombardi paper differ among themself in the outcome of XMRV+ CFS patients:

a) nested gag PCR 67% (68/101)

b) protein expression 63% (19/30)

c) that is not easy to figure out for PBMC-LNCaP transmission, because there is no number of patients given and there are different patient ID numbers used in Fig. 3A and Fig. S5. For plasma-LNCaP transmission they found 83,3% positive.

d) antibody response 50% (9/18)

So talking about 67% positive is the result of PCR alone (nested - according to the text, single round - according to figure legend, activation of PBMC before PCR was not mentioned), like done in the european studies.

Permalink to Comment

51. Camaro on January 19, 2011 9:23 AM writes...

Sorry, forgot numbers for plasma-LNCaP transmission : 83,3% (10/12)

Permalink to Comment

52. RRM on January 19, 2011 12:30 PM writes...

@Anonymous

Anonymous--> "Correct, as it was the result of using multiple technologies:

Again, can you point me to a line in the original study that asserts or implies this? In the Science addendum (published in Landesbioscience), Mikovits even stated that: "Of the 34 patients whose PBMCs were negative for XMRV by DNA or cDNA PCR, 17 were positive for infectious virus when co-cultured with the LNCaP indicator cell line..." Which means she still agrees that that a whole lot of the Science samples (I guess she just miscounted 1 patient because of the 67% percentage) tested positive for "just PCR" in some way or another.

But even hypothetically accepting my misunderstanding of that line and accepting you assertion as fact, the argument makes no real sense. In the already mentioned Science addendum Mikovits still agreed that the 7 out of 11 patients that were PCR positive for both gag and env on figure 1A of the Sciende paper, were tested using just (single round) PCR without culturing.

Bottom line: Even though Mikovits also stated that these samples were actually a subset of patients with persistent viremia (again, see the addendum), these PCR-only results should be easily reproducible by Mikovits or others, using "just PCR". Conclusion: she is implicitly retracting her earlier findings when she is stating that 'she is not into PCR' and that 'she was just asked to do this unreliable PCR thingy for the BWG'.

Anonymous-->"RRM in response to Hohn et al. did say that Lombardi et al. used the same kit"

Sigh. Literal copy/paste from that discussion:

RRM says (caps added for emphasis) "Sato et al did not test the kit that Lombardi et al or Lo et all used. sato et al, however, did test A KIT FROM THE SAME BRAND that was used by Lombardi et al. They found that kit (I believe out of a total of 4 kits) to be contaminated."

I can not understand how, given the above quote, you seem to question my earlier remark.

Anonymous-->"The negative papers are looking at general fatigue or even chronic fatigue, AKA CFS."

This was not directed to me, but I just want to say that this is being repeated ad nauseum on internet forums, but that doesn't make it true. Mikovits was sent at least 20 samples from the


Permalink to Comment

53. Anonymous on January 19, 2011 1:13 PM writes...

Do you have a line that says different?

Mikovits and those who collaborated on Lombardi et al. have always stated that PCR alone is the least sensitive technique, and that 7 of 11 patients exhibited the expected gag and env PCR amplification products from single-round PCR with XMRV primers. Those patients had persistent viremia. Lombardi et al. even states "Detection of XMRV was confirmed in 7 of 11 WPI CFS samples at the Cleveland Clinic by PCR-amplifying and sequencing segments of XMRV env [352 nucleotides (nt)] and gag (736 nt) in CFS PBMC DNA (Fig. 1A)" Only the data from those 11 patients, and 11 controls are shown for single round PCR.

Other labs have not matched Lombardi et al's methodology for even single round PCR. None have used the Canadian criteria, many have used different primers, all negative papers have used previously processed blood - not blood collected the same way as Lombardi et al. All negative papers have calibrated to a clone, and not a positive clinical sample, which greatly limits the detectable diversity of the virus. There's a hugh giant list of differences. In conclusion Mikovits, Ruscetti and others have never in any way retracted their earlier findings. And no the WPI does not and has never done direct PCR, read the paper again.

Do you know why that one patient was not included in the figure?

You did say Lombardi used the same kit, and they did not. It is irrelevant if they were contaminated, if they used a different kit and checked for contamination.

Funnily the CDC disagreed with you about who the Science paper was studying. ME/CFS is not CFS and never has been. Time you learnt to live with that.

Permalink to Comment

54. Anonymous on January 19, 2011 1:15 PM writes...

Do you have a line that says different?

Mikovits and those who collaborated on Lombardi et al. have always stated that PCR alone is the least sensitive technique, and that 7 of 11 patients exhibited the expected gag and env PCR amplification products from single-round PCR with XMRV primers. Those patients had persistent viremia. Lombardi et al. even states "Detection of XMRV was confirmed in 7 of 11 WPI CFS samples at the Cleveland Clinic by PCR-amplifying and sequencing segments of XMRV env [352 nucleotides (nt)] and gag (736 nt) in CFS PBMC DNA (Fig. 1A)" Only the data from those 11 patients, and 11 controls are shown for single round PCR.

Other labs have not matched Lombardi et al's methodology for even single round PCR. None have used the Canadian criteria, many have used different primers, all negative papers have used previously processed blood - not blood collected the same way as Lombardi et al. All negative papers have calibrated to a clone, and not a positive clinical sample, which greatly limits the detectable diversity of the virus. There's a hugh giant list of differences. In conclusion Mikovits, Ruscetti and others have never in any way retracted their earlier findings. And no the WPI does not and has never done direct PCR, read the paper again.

Do you know why that one patient was not included in the figure?

You did say Lombardi used the same kit, and they did not. It is irrelevant if they were contaminated, if they used a different kit and checked for contamination.

Funnily the CDC disagreed with you about who the Science paper was studying. ME/CFS is not CFS and never has been. Time you learnt to live with that.

Permalink to Comment

55. Anonymous on January 19, 2011 1:17 PM writes...

Do you have a line that says different?

Mikovits and those who collaborated on Lombardi et al. have always stated that PCR alone is the least sensitive technique, and that 7 of 11 patients exhibited the expected gag and env PCR amplification products from single-round PCR with XMRV primers. Those patients had persistent viremia. Lombardi et al. even states "Detection of XMRV was confirmed in 7 of 11 WPI CFS samples at the Cleveland Clinic by PCR-amplifying and sequencing segments of XMRV env [352 nucleotides (nt)] and gag (736 nt) in CFS PBMC DNA (Fig. 1A)" Only the data from those 11 patients, and 11 controls are shown for single round PCR.

Other labs have not matched Lombardi et al's methodology for even single round PCR. None have used the Canadian criteria, many have used different primers, all negative papers have used previously processed blood - not blood collected the same way as Lombardi et al. All negative papers have calibrated to a clone, and not a positive clinical sample, which greatly limits the detectable diversity of the virus. There's a hugh giant list of differences. In conclusion Mikovits, Ruscetti and others have never in any way retracted their earlier findings. And no the WPI does not and has never done direct PCR, read the paper again.

Do you know why that one patient was not included in the figure?

You did say Lombardi used the same kit, and they did not. It is irrelevant if they were contaminated, if they used a different kit and checked for contamination.

Funnily the CDC disagreed with you about who the Science paper was studying. ME/CFS is not CFS and never has been. Time you learnt to live with that.

Permalink to Comment

56. Camaro on January 19, 2011 1:47 PM writes...

@Anonymous above

"All negative papers have calibrated to a clone, and not a positive clinical sample, which greatly limits the detectable diversity of the virus."

Please explain why this should be a problem, as the sequences found by Lombardi et al were i) found with primers based on the VP62 molecular clone sequence and ii) more than 99% identical to the BP35, VP42 and VP62 clones (see Fig S1).

Permalink to Comment

57. Anonymous on January 19, 2011 2:08 PM writes...

Obviously it is a problem if you have a person with XMRV that is outside the diversity of the clone. Never mind the P and M variety also identified by other labs. More on this will be published soon. Hence why the use of multiple methods are also needed. Lombardi et al. employed only 30 base pairs, this is now up to 700 and rising.

Permalink to Comment

58. Anonymous on January 19, 2011 2:10 PM writes...

Partly Lombardi et al. was lucky, if not also talented.

Permalink to Comment

59. Wondering on January 20, 2011 1:13 PM writes...

Hi I was wondering after reading some of the posts above what you all make of the fact that in the Science study only about 6% of the healthy controls were positive. If contamination of the test kits was a major issue wouldn't that number have been higher too?

Permalink to Comment

60. Anonymous on January 20, 2011 2:36 PM writes...

Yes, contamination would have been similar in both groups if it was contamination. The contamination argument is very weak.

Permalink to Comment

61. Eco on January 21, 2011 3:13 PM writes...

I wish our esteem colleague ERV or Abbie 0f Erv scientific Blog post would invest herself into the FDA Blood Products Advisory Committee so as to save humanity and taxpayers dollars from being wasted for investigating a non existent virus at worse or a contaminate at best. Please ERV with your eminent knowledge on qPCR techniques that you stated in one of your blogs that even an graduate student should know the basic techniques of using.

Please inform and teach the following doctors on the basic techniques of qPCR assays and contamination as apparently they lack the fundamental knowledge, education and skill set behind the science on this technique.
Dr. Coffin, Dr. Alter, Maureen Hanson,
Dr. Celso Bianco, Dr. Hollinger, Dr. Judy Mikovits, Dr. Jonathan Stoye, Dr.Francois Villinger, Dr.Shyh-Ching Lo, Dr. Racel Bagni, Dr. Epstein, Dr. Nelson, Dr. Klimas, Dr. Demetriades, Dr. Gylnn, Dr. Monroe, Dr. Bianco, Dr. Katz, Dr. Kleiman, as well as such organizations as the NIH, FDA, American Red Cross, ABBA.

as well as outside the organization. Dr. Singh, Dr. Dusty miller, Dr.Vincent Racaniello, Dr. Jolicoeur, Dr. Houghton.Dr. Lorne Tyrrell,Dr. Eleanor Stein etc.

If you could point them to one of your published peer review articles on the subject, I am sure they will be most indebted to you explanation why they are wasting our taxpayer's money on the subject. Please set them straight on this issue.

Permalink to Comment

62. Eco on January 21, 2011 3:23 PM writes...

I am astounding and quite impressed on ERV's analysis of the situation. She always brings such incredible insight and assessment to the issue of XMRV. I am sure the University of Oklahoma faculty staff is proud to have her as one of their graduate students.

With that being said, I wish our esteem colleague ERV or Abbie 0f Erv Scientific Blog post would invest herself into the FDA Blood Products Advisory Committee so as to save humanity and taxpayers dollars from being wasted for investigating a non existent virus at worse or a contaminate at best. Please ERV with your eminent knowledge on qPCR techniques that you stated in one of your blogs that even an graduate student should know the basic techniques of using.

Please inform and teach the following doctors on the basic techniques of qPCR assays and contamination as apparently they lack the fundamental knowledge, education and skill set behind the science on this technique.
Dr. Coffin, Dr. Alter, Maureen Hanson,Dr. Celso Bianco, Dr. Hollinger, Dr. Judy Mikovits, Dr. Jonathan Stoye, Dr.Francois Villinger, Dr.Shyh-Ching Lo, Dr. Racel Bagni, Dr. Epstein, Dr. Nelson, Dr. Klimas, Dr. Demetriades, Dr. Gylnn, Dr. Monroe, Dr. Bianco, Dr. Katz, Dr. Kleiman, as well as such organizations as the NIH, FDA, CDC, NCI, The American Red Cross, & ABBA.

As well as outside the organization. Dr. Singh, Dr. Dusty Miller, Dr.Vincent Racaniello, Dr. Jolicoeur, Dr. Houghton, Dr. Lorne Tyrrell, Dr. Eleanor Stein, Dr. Silverman, Dr. Klien, Dr. Ruscetti, etc.

If you could point them to one of your published peer review articles on the subject, I am sure they will be most indebted to you explanation why they are wasting our taxpayer's money on the subject. Please, Abbie, set them straight on this issue as we need to fund more worthwhile research projects.

Permalink to Comment

63. RRM on January 23, 2011 3:03 AM writes...

@Anonymous

Anonymous: "You did say Lombardi used the same kit, and they did not."

I have provided a literal quote for my argument, while you continue to post a statement without backing it up. I suggest you provide a quote to support your (untrue) assertion or concede the point.

Anonymous: "Mikovits and those who collaborated on Lombardi et al. have always stated that PCR alone is the least sensitive technique"

Still, their PCR results should be REPRODUCIBLE in itself.

The scientists from the Science paper have unequivocally stated that their 67 percent result came from performing nested PCR for gag sequences. Without culturing. It isn't very useful to point out how other methods might be more reliable, because this a published result that should be reproducible in itself.

Anonymous: "All negative papers have calibrated to a clone, and not a positive clinical sample, which greatly limits the detectable diversity of the virus""

Have you ever heard of a ad hoc hypothesis? I suggest a quick google search.

By the way: there are no positive clinical samples, only samples that are positive according to one lab but that fail to be recognized by that one lab in the blinded setting of the Blood Working Group.

Anonymous: "And no the WPI does not and has never done direct PCR, read the paper again."

Yes, I have read the paper and yes, the WPI have performed "direct" nested PCR for gag on uncultured samples. The WPI found an impressive 67% of patients positive for XMRV gag sequences.

I suggest you read the original paper, the press release, and the addendum again if this is not clear.

Anonumous: "Do you know why that one patient was not included in the figure?"

No. Please share.

(Perhaps another) Anonymous: "Yes, contamination would have been similar in both groups if it was contamination. The contamination argument is very weak."

No, it is not as, there are several precedents for these kinds of results.

Even now, WPI are agreeing that experiments should be done to rule out contamination in future studies (see their 1/4 press release). I guess you find the WPI and its scientists of subpar quality for advancing this "very weak argument"? After all, why check for contamination when different results between patients and controls already conclusively disprove contamination in itself?

Permalink to Comment

64. Camaro on January 24, 2011 3:24 AM writes...

@Anonymous, post #57 + #58

Obviously I have to say it again; Lombardi published only clonal sequences, therefore advising all other labs that finding XMRV is difficult due to the diversity seems a bit strange.

Another thing concerning the use of “PCR only�, remember the Lo/Alter paper was very welcomed by the WPI:

http://www.wpinstitute.org/news/docs/WPI_pressrel_082310.pdf


First of all, the paper is called “replication study� despite the fact that the only method used is nested PCR on uncultured samples.

Second, and let’s be clear about this, the Lo/Alter group did NOT find XMRV, but there was no published advice to go back and do the other couple of methods until you found “the real XMRV�.

Permalink to Comment

65. pianoplayrr on March 2, 2011 4:00 PM writes...

I'm only a simple engineer, having studied both the Chemical and Electrical disciplines. But my wife has been ill with, what has been characterized as, CFS. Coming at this from another angle, any way you choose to look at it, SOMETHING is causing this. This was a educated, determined woman barely 50, wife, mother, who worked for almost 30 years as a psych. nurse, raised 3 children, and who is now housebound, confused, in unending pain, severely depressed, and almost constantly beddridden from extreme fatigue. No matter what the hypothesis, more people appear to have this than do those with AIDS. If there is even a 10% chance that is of viral origin, TIMES a 10% chance that it is (or may mutate into) an easily tranmissable pathogen, this means that - given the dynamics of past epidemics and the present day ease of world travel, then the entire planet is potentially at risk. (30 Million people died in 1918 from Spanish Flu - before the Jet Age!) People with CFS dont just "die", rather they waste away over a period of years. My proposition may not be very scientific, but should alarm us enough to press our government to commit resources to resolving this. So far, we've spent more time and $$ on publicity alone for the Swine Flu/H1N1 and Ebola "scares" combined!

Permalink to Comment

66. Gob on March 10, 2011 8:15 PM writes...

Monoclonal antibody to SFFV env will only react with the SU protein of a MLV virus. There were no other MLV viruses in the experimental environment with the same genomic sequence as XMRV. Would you care to explain what a monoclonal antibody to SFFV env could cross react with and do you have a reference please?

Lo et al confirmed the findings of Lombardi et al. XMRV is a Polytropic/Xenotropic hybrid.

All the negative studies have used the VP62 clone.

Permalink to Comment

67. XMRV + since 2010 on March 25, 2011 7:41 AM writes...

Brain Demyelination/lesions AIDS Yes ME/CFS Yes

Chronic sore throat Flu like illness AIDS yes ME/CFS yes

Swollen lymph nodes AIDS yes ME/CFS Yes

Cognitive problems AIDS yes ME/CFS yes

Skin Problems AIDS yes ME/CFS yes

IBS and other stomach problems AIDS yes ME/CFS Yes

Cancer/Leukimia/Lymphoma AIDS yes ME/CFS Yes

Weird tumor
spleen,liver,brain,testicular AIDS yes ME/CFS yes

Heart Failure AIDS yes ME/CFS yes

Many active viruses AIDS yes ME/CFS yes

White thrush AIDS yes ME/CFS yes

Swollen tongue with teethmarks around it AIDS yes ME/CFS yes

Fatigue AIDS yes ME/CFS yes

Seizures AIDS yes ME/CFS yes

How blind and gullible do you have to be, to not be able to see that ME/CFS is also caused by an HIV like virus? ME/CFS is caused by a retrovirus, we have known this since the early 80s when ME/CFS brain MRIs were taken and they looked exactly like AIDS patients, in the early 90s De Freitas found a retrovirus in the blood of ME/CFS patients and the CDC didn't even bother paying attention to her, now the WPI has found the XMRV virus which is a retrovirus which was suspected all along and people still can't admit that ME/CFS is caused by just that.,

How blind and dumb are you?

How can you tell me that is all in my head ?when two of my friends that have HIV have the very same health problems that I do but they do much much better then me because they have meds and I don't.

Permalink to Comment

68. luysii on March 25, 2011 3:01 PM writes...

Have a look at Science vol. 331 pp. 1253 - 1254 '11 (11 March) in which evidence is presented that researchers essentially inadvertently CREATED XMRV while passaging human prostate tumor samples in mouse cell lines. This is very creepy for us all, CFS or not, as the virus readily infects human cells. It's fairly technical but certainly worth a read.

Is anyone else having problems receiving a print subscription to Science? Here it is the 25th and the 11 Mar '11 Science is the latest one i have.

Permalink to Comment

69. ND on July 2, 2011 11:49 AM writes...

Paprotka et al. is flawed. Science should retract it.

Read this

http://www.imeassoc.com/Response__Paprotka_et_al.html

Permalink to Comment

70. Emily A. Cox on June 13, 2012 10:10 PM writes...

Holy shit, this person's story:

"Back in May 5th 2010 i was sexually involved with a girl and when we were doing it i noticed the condom had broken when i withdrew my first thought was "i hope i don't get this girl pregnant" but she told me she was on the pill so i went on with my life,(all these following symptoms i have never seen or felt before in my life) two weeks went by and i had a couple of hot flashes but didn't make a big deal of it, two more weeks went by and i noticed i had many many little pimples on my back right behind my armpits around this same time i started to feel confused but i thought maybe i didn't get enough sleep, around the 5th week i started to get worried because i was feeling nauseated and somewhere around the 6th week i had a "viral spike" i got really sick with all sorts of symptoms, nausea,confusion,weakness, constipation,fatigue, night sweats, lost like 15lbs in a few days, i had a really bad rash on my back.

i thought for sure i had gotten HIV, i went to get tested for HIV on July 21, negative."

This is EXACTLY my story! I too was sexual with a woman that I was dating (I am a woman, too), and I sucked her breast and she lactated and some blood came out of her breast... it was so strange, which we both commented on because she said she was "not pregnant." She at that time, by the way, claimed that she had "not been feeling well..." just extremely "tired," and had one tonsil that was enlarged and asymptotical. This incident between us occurred twice... one on July 21st 2008 and the second time on August 22nd 2008. I do not remember which one where she lactated or bled, but it was one, or one was the 21st and the other the 22nd. Irregardless, I became very sick the last week of August. Between July 22nd and the 2nd incident on August 22nd, I had traveled back home 20 hours away, and when I arrived back home, I thought I had maybe picked up something from family or simply worn myself out over the travels... Definitely not.

I ended up with hot and cold sweats, like DRENCHING, major EAR pain, which I would describe as "ear/jaw" pain (No, NOT TMJ), joint pain, shoulder pain that burned like you wouldn't believe, swollen lymph nodes, weakness, etc. I thought I had strep throat, but didn't. Things got worse... worse worse. To make a long story short, I too, thought I might have contracted HIV or some other STD and got tested MULTIPLE times, from MULTIPLE facilities, and dr's began to tell me it was "all in my head," and I was a "hypochondriac," etc. but I was paranoid and SURE I had contracted HIV. I even wrote a fear-based nasty email to this past girlfriend of mine saying, "I think you 'might' have given me HIV and it hasn't shown up yet." Nope, it's CFS. Or well, I am almost positive that it is.

I found the link between all of my symptoms, etc. in 2010 online when XMRV just came out or was discovered, before anyone. It's actually in/on my online journal... anyway, I have been diagnosed with lyme disease by Igenix, also.

There is so much more, and I have tons and tons of documentation, etc. but I won't share everything on here due to time constraints. I just wanted to say that this individual's story is almost IDENTICAL to mine.

If anyone wants to talk about this, or if there are any researchers who want to contact me, please email me:

Not2bforgot10@yahoo.com

That's "Not 2 b forgot 10" at yahoo dot com

Permalink to Comment

POST A COMMENT




Remember Me?



EMAIL THIS ENTRY TO A FRIEND

Email this entry to:

Your email address:

Message (optional):




RELATED ENTRIES
XKCD on Protein Folding
The 2014 Chemistry Nobel: Beating the Diffraction Limit
German Pharma, Or What's Left of It
Sunesis Fails with Vosaroxin
A New Way to Estimate a Compound's Chances?
Meinwald Honored
Molecular Biology Turns Into Chemistry
Speaking at Northeastern