XMRV and chronic fatigue syndrome: A Tale of Two Three Laboratories

A couple of updates that might add more to the discussion:

1) WORLD RENOWNED RETROVIROLOGISTS SOUND OFF ON VAN KUPPEVELD ET AL, BRITISH MEDICAL JOURNAL XMRV ETHICS AND SCIENCE
Below are the reactions of two of the world's most eminent retrovirologists - quoted at this week's Prague 2010 Centennial Retrovirus Conference in an article entitled, "Positive XMRV Study a Matter of Time". The following quotes address the withholding by van Kuppeveld et al and the British Medical Journal, of their positive test results conducted by the Whittemore Peterson Institute; the rumors circulated by the Nijmegen team (van Kuppeveld et al) that contamination was an issue in the Science team's work; and the apples-to-oranges situation in which none of the rebuttal studies used all 4 techniques that the Science team did:

DUTCH PRESS RELEASE : POSITIVE âXMRV-STUDYâ A MATTER OF TIME
Prague - May 3, 2010. Last October U.S. scientists presented a breakthrough around the research on chronic fatigue syndrome (CFS), which was published in Science. They found traces of the retrovirus XMRV in the blood of CFS patients. Thereafter, three groups of European researchers, including a Dutch group from Nijmegen, couldnât confirm these findings. However, at the 'Centennial Retrovirus Meeting' in Prague it became clear that the first positive 'replication study' seems only a matter of time.

The June issue of the Dutch magazine Ortho will focus on the multi-day conference in Prague, which ends today. Especially in the corridors this new retrovirus was the talk of the day. Insiders agree that the negative XMRV-studies which have been published so far, were not exact replication studies. The several groups of researchers used techniques that differed too much from those used by the U.S. researchers. This is also true for a yet unpublished German study, where XMRV wasnât found in blood samples from CFS patients either.

Recently the American scientist Dr. Judy Mikovits visited several European research groups to help them with the proper laboratory technique. It is now clear that these visits are starting to pay off. During the Prague Conference Mikovits explained once more in great detail the complex methodology of the Whittemore-Peterson Institute (WPI), the National Cancer Institute (NCI) and the Cleveland Clinic. This methodology of culturing the virus is imperative because XMRV is only present in extremely low concentrations in the peripheral blood.

Dr. Francis Ruscetti of the renowned NCI â an U.S. government agency â told Ortho that he hopes this controversy will all die away in 2011. He is especially surprised about the fact that the investigators of the UMC St. Radboud at Nijmegen concealed in their publication that the Americans found traces of XMRV in the same blood samples from the Dutch patients. "I donât know how they get away ethically with this," said Ruscetti. "I donât think that is good science." Ruscetti pointed out again that the WPI, the NCI and the Cleveland Clinic applied four procedures in their research. "In those negative studies they only tried one." Ruscetti also ventilated his annoyance over what he calls the "whispering campaign" about contamination. According to the Nijmegen researchers, the Americans contaminated or polluted the Dutch blood samples.

AMONG OTHERS, RUSCETTI IS SUPPORTED BY PROF. DR. JOHN COFFIN, who is linked both to the NCI and Tufts University in Boston. He is considered one of the most prominent retrovirologists in the world. "People have raised the issue of contamination," said Coffin. "But we donât really know anything about that yet. We donât have any evidence. A lot of the studies were done in fact at the NCI, in the lab of Francis and Sandra Ruscetti. They have a long experience with these viruses and are very carefull workers."

Coffin emphasized once again that doing a replication study implies that it is performed in exactly the same way. "In none of the studies that have been published so far that were negative, the virus was cultured," said Coffin. "Only the Science-study did this, which is a very compelling point."

Researchers from Nijmegen were not present at this leading conference.
English Translation by European Society for Myalgic Encephalomyelitis:
http://esme-eu.com/news/dutch-press-release-positive-xmrv-study-a-matte…
Original article in Dutch:
http://www.orthoeurope.com/persberichten/95/positieve-xmrvstudie-kwesti…

2)ABOUT VAN KUPPEVELDâS REBUTTAL LETTER (http://www.umcn.nl/Research/Departments/kcv/Documents/F%20v%20Kuppeveld…) In this open letter to the WPI, Dr van Kuppeveld confirms that samples of his cohort were sent to the WPI and did test positive - but his team intentionally withheld this information:

"Given the robustness of our paper, we considered it scientifically premature to report this finding before having settled the reason for the discrepancy." Interestingly, van Kuppeveld also notes that the paper was rejected by the Lancet.

Yet the BMJ published this work? Did or did not the BMJ know about this withholding of information? And isn't that what the Discussion section of research papers is for? Since when is it OK to not publish conflicting results, and to assume that you â rather than the readers of the BMJ - know the reason for the discrepancy? Itâs a little like saying you have better information than the market, in stock investing.

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I'm hanging my hat with Coffin, Ruscetti et al. - not a bunch of psychiatrists and their cabal, who stand to lose their reputations, fiefdoms, contracts with insurance and pharma companies, and more. And not, I might add, with an invective-laden trigger-happy blogger who has about the same degree of intellectual curiosity as Sarah Palin. Yes, the esteemed NCI researchers may prove that XMRV isnât âitâ in ME/CFS (tho I doubt it). But at least they will have done so thoroughly and fastidiously: thatâs all patients ask for. In the interim however, the noses of XMRV research wannabeâs are definitely out of joint at an upstart, and innovative grassroots enterprise that is supremely motivated to find a cure - albeit on a learning curve when it comes to PR. It is nothing short of blasphemy to the establishment to have an upstart medical centre leap onto the international stage with a publication in Science that sets psychiatric flat-earthers straight on the third human retrovirus.

All this to say - keep an open mind on the emerging XMRV/ME/CFS science, and for those of you also digging into analysis, DO also consider whether we are comparing apples with oranges - and why. Would you do research on appendicitis, using cohorts that included patients with diffuse stomach ache, premenstrual cramps, and stomach flu? No wonder muddied ME/CFS cohorts result in botched XMRV studiesâ¦.

Now stay tuned for major revisions â or outright retraction of the BMJ van Kuppeveld paper.

Coffin, credulous? ERV, a genius? You've got it backwards. If you posters here would talk to someone besides yourselves you might get a more accurate picture.

McClure's the idiot. She did Simon Wessely's dirty work and now it's come back to bite her in the butt. ZERO XMRV in UK? Give me a break! She was 1000% sure, after a 3 week study? Right! Her radio interview in Australia showed she now knows she was set up.

Point 1 in Kuppeveld's letter is in error, if not a lie. If an apology is owed, it is from him, not Ruscetti. Maybe his colleague has a problem with the English language. It has been known from the beginning that the WPI samples did not come from an "outbreak", but McClure et al keep repeating this, even after being informed otherwise. This is just more smoke to keep anyone from really looking into XMRV. "Lab contamination" was laid to rest long ago, but they think that if they chant it often enough, dumb bunnies will believe. I guess it worked for ERV and her little fan club.

The "real deal" about the Dutch samples is that they were incredibly old (why?), they came from self-diagnosed patients with fatigue, which is not CFS or ME, over a third were on antidepressents, which fits perfectly with their Oxford definition which was concocted by psychiatrists in order to capture ME/CFS for their own benefit and turn it into a mental illness.

This is what comes of allowing psychiatrists to control research into biomedical illnesses. At least in the US, retrovirologists and other real scientists have taken back the field from the psychiatrists. Any retrovirologist or other real researcher worth her salt ought to be applauding that instead of shoveling the BS on behalf of the psychiatrists.

found this on youtube,

http://www.youtube.com/watch?v=iWvWs1rGhrE

erv says "She also doesnt mention that extra-special 'culturing' Ive heard mentioned before"

Presumably the sections labelled
"2-VIRAL AMPLIFICATION BY TRANSMISSION TO LNCaP (Biological amplification)"
and
"3-VIRAL ISOLATION"

will be culturing. Or didn't they teach that isolation=culturing in your school?

I love the Dutch. They raise casual indifference to the level of high art.

I'm curious how you got the letter. I would assume that their professional response would be a private communication.

What's that part about "we suspected contamination of our samples in your [Whittemore's] laboratory"?! It sounds like the Dutch group sent some samples for WPI to test, and that WPI found 2 positives in 7 CFS samples vs. 1 positive in 3 control samples. Yet the Dutch could not confirm any of those positives in their own lab, using the sample samples!

Is this new info? I haven't seen any previous reference to it. However, there is this bit from Whittemore's letter to Dr. McClure:

In addition, Dr. van Kuppeveld asked for and received reagents and a positive patient sample to determine if his testing procedures could in fact detect XMRV in a positive blood sample before he published his paper. We wonder why these materials were not used in his study which also failed to detect XMRV.

If I'm understanding this correctly, this is as close as anyone's yet come to showing that WPI's results are due to lab contamination.

I think the WPI came very close to it themselves when they reported their sequences to NCBI NucCore. How very bizarre that the sequences of retroviruses hanging out in outbreak patients since 1984 are virtually identical to the positive control from Silverman.

Strange that both sides are claiming that they wanted to swap samples, and the others are not replying.

More letters.

McClure to WPI:
https://www.wpinstitute.org/news/docs/mclure-wpi_ltr_apr10.pdf

Reply from WPI:
https://www.wpinstitute.org/news/docs/wpi-mclure_ltr_apr10.pdf

Funny article/ press release that portrays Francis Ruscetti and John Coffin as rather supportive of the WPI:
http://esme-eu.com/news/dutch-press-release-positive-xmrv-study-a-matte…

I'm always a bit suspicious of this sort of quoting and paraphrasing though.

There are rumors of a couple more negative studies coming soon, and of positive (?) studies being expanded. I think that this is going to go on for a fair bit longer.

Here is WPI's exact methodology. http://www.iacfsme.org/BULLETINSPRING2010/Spring2010MikovitsLetter/tabi…

Either way giving this level of detail is going to bring this to a head one way or the other.

Also read this quote... Are Coffin and Ruscetti consider quacks as well?

Dr. Ruscetti, one of the co-authors of the original study, evinced considerable frustration as he let loose with a blast probably rarely seen in the staid retroviral world calling the Dutch researchers burial of contrary results unethical and poor Science to boot ""I do not know how they think they get away with this ethically," said Ruscetti. "I do not think this is good science." Dr. Mikovits said she'd never seen anything like it and Dr. Ruscetti apparently never has either. He noted, as Dr. Mikovits repeatedly has, that the WPI, NCI and Cleveland clinic tested XMRV in four different ways and all checked out. The negative studies only looked for XMRV in one way (PCR) using a methodology that both researchers felt was problematic. Dr. Ruscetti also expressed his dismay about what he called a 'whisper campaign' about contamination.

Dr. Coffin, a top retrovirologists, strongly defended the XMRV finding. In a translation of a Dutch report Dr. Coffin reportedly said "People have raised the issue of contamination but nothing is known about at the moment. There is no proof. Much of the research is done at the NCI, in the laboratory of Francis and Sandra Ruscetti. They have a long experience with these viruses and are very cautious." Indeed, both of the Dr's Ruscetti are long-time employees of the National Cancer Institute - one of the most highly thought of institutions in the world. Dr. Coffin also noted a key difference between the original Science paper and the 3 validation studies; the fact that only the WPI study has 'grown' the virus:"None of the negative studies have been published so far, the virus is grown," said Coffin. "Only in the Science study has been done, and that is a very strong point."

qetzal-- :D

gf1-- McClure is a kick ass lady.

Impish-- I think Coffin and Ruscetti were lied to by Judy and Annette, either purposefully or because those two women are fantastically stupid. In any case, I do expect Dr. Ruscetti to send an apology to Dr. van Kuppeveld, now that he knows the real deal with the Dutch samples.

Coffins completely credulous take on XMRV-->CFS has been a point of discussion among some of us. Its odd.

@ ERV: Did you have a look at the 'How to find XMRV the WPI way' piece Impish posted?

Does its virology make any sense? Could it help explain the differing results? It could be complete nonsense, and I don't think I'd be able to tell. Thanks.

Isn't Ruscetti Judy's former boss? She was a technician, grad student, and post-doc at NCI, was she with him the whole time or just part of it?

http://imedia.unr.edu/cmm/cmm.html

Hunh. No steeple. No stained glass. No pews. How is anyone supposed to know that you go there to tithe and worship?

gf1-- The protocol looks completely dull and uninteresting.

I have invented several things in the lab which have required specific tips/tricks that people might not think to do, to make the system work ie, you have to have the annealing time extended up to two minutes, not 15-30 seconds, need 5 times as much forward primer in the second round of PCR to get the size product you want, need to clone using a double digest as sequential will not work, you have to let the fluorescent protein mature for 5 days, not 2).

There are any number of 'weird' things that you sometimes have to do to make stuff work in the lab. When you have to do weird stuff, you make a point of putting that in your published protocol, or else no one can duplicate your results.

There is nothing weird in that PCR protocol.

I wouldnt have used a random oligo in the RT, since you know what you are looking for, and I think they added too much DNA into their PCR (Ive found theres not much of a point to adding more than 100 ng with Real-Time, otherwise your just increasing your background), theyre also adding about twice as much primer as I do, but, meh.

Not enough detail for their second round of PCR for me to judge it. Second round PCR can be tricky.

But again, there is nothing weird (ie, 'We had to anneal at 54 C and do a 0.2 touch-down' or 'We had to do a 2 minute extension, even though its only a 500 bp fragment' or 'We had to do 50 cycles, not 35'). She also doesnt mention that extra-special 'culturing' Ive heard mentioned before.

There is no reason why this very standard PCR should be so 'difficult' for other labs. There is no reason why this protocol should be considered super/special/magic.

Sandy-- heh. Good for her. Bossman would kick my ass if I was pulling her shit (technically, Bossman is a stickler for controls and always being 100% sure-- a perfectionist. I am too. we wouldnt have gotten into this mess in the first place).

Thanks for that ERV. I was surprised that it didn't seem to mention the culturing that others had said was important (I thought the WPI had said this too).

I'm also a bit surprised they said "despite the current assumption that PCR answers all questions, there are serious flaws in this assumption" - surely that misses the point. If their PCR tests are picking up an XMRV/CFS correlation, and no-one elses are, that's a problem regardless of their other testing.

If their immune response tests are less specific, maybe they've found something other than XMRV? But then, they're meant to have sequenced whatever they found and confirmed it was. (Pardon the surely garbled terminology here).

Science has published 3 comments from other scientists and a response by Mikovits and Ruscetti. The links can be found in this note:

http://www.facebook.com/notes/the-cfids-association-of-america/more-on-…

We await your commentary. :)

Wow to the Science letters, Ruscetti's going all the way for Mikovits. Silverman, Peterson and the rest didn't sign the response, they stayed out of it. Good choice. Changing stories, inaccurate language, and we still don't know where damn samples came from. Or which local medical practice provided the controls. You know, "You stupid Dutch people didn't understand my presentation" isn't an effective response to criticism of holy-shit jaw dropping sloppiness. Or classy, but we didn't expect that from Judy.

I am not a scientist so ERV would be much better qualified to respond to the details of this but I am annoyed by several pieces of information in these letters. I am busy today so I will do these one at a time. The first one is thisâ¦

In the letter from Wessely they state âCFS is likely to arise from complex genes-x-environment risk factors, making a simple causative link between XMRV and CFS unlikely.â
Seriously⦠You have the balls to write in a scientific journal in a response outlining how you think someoneâs science is bad a statement like that with absolutely nothing backing it up.

I think that x is true even though I have no proof so that obviously means that your assertion that y is true is false. Wowâ¦

On the other hand, WPI is full of crap whtn they say that they don't want to raise false hopes. They are all over the place basically saying XMRV = CFS. Many of the people who suffer from this now have a great deal of hope put into the link between XMRV and CFS largely due to the publicity efforts of this lab. If this turns out to be false they should be tarred and feathered.

On the other hand a bunch of the scientists on the other side have been advocating Cognative Behavioural Therapy and gradual increasing exercise as a treatment for what is clearly a physical disease. This continues even though a recent (too lazy to cite at the moment) study showed absolutely no positive effect on patients this treatment is given to. Literally millions of dollars have gone into this form of non-treatment as well as a good deal of the CFS research dollars in Europe.

If it turns out not to be XMRV hopefully this whole thing will cause someone to figure out what it actually is as it certainly is not a mental illness.

Maybe ERV will find it :-)

Regardless of your opinion of the WPI, it looks like the WPI, Cleveland Clinic and and the NCI are not alone any longer. The CDC expedited this for the June issue:

Xenotropic Murine Leukemia Virusârelated Gammaretrovirus in Respiratory Tract

http://www.cdc.gov/eid/content/16/6/pdfs/10-0066.pdf

3% XMRV+ in respiratory secretions for the controls.
10% XMRV+ in immuno-supressed transplant patients.

http://www.cdc.gov/ncidod/EID/index.htm

Perhaps XMRV can swim.

Why do you think that paper has anything to do with WPI, or their claims on the association between XMRV & CFS?