Monday, 20 December 2010

XMRV - Innocent on All Counts?

A bombshell has just gone off in the continuing debate over XMRV, the virus that may or may not cause chronic fatigue syndrome. Actually, 4 bombshells.

A set of papers out today in Retrovirology (1,2,3,4) claim that many previous studies claiming to have found the virus haven't actually been detecting XMRV at all.

Here's the rub. XMRV is a retrovirus, a class of bugs that includes HIV. Retroviruses are composed of RNA, but they can insert themselves into the genetic material of host cells as DNA. This is how they reproduce: once their DNA is part of the host cell's chromosomes, that cell is ends up making more copies of the virus.

But there are lots of retroviruses out there, and there used to be yet others that are now extinct. So bits of retroviral DNA are scattered throughout the genome of animals. These are called endogenonous retro-viruses (ERVs).

XMRV is extremely similar to certain ERVs found in the DNA of mice. And mice are the most popular laboratory mammals in the world. So you can see the potential problem: laboratories all over the world are full of mice, but mouse DNA might show up as "XMRV" DNA on PCR tests.

Wary virologists take precautions against this by checking specifically for mouse DNA. But most mouse-contamination tests are targeted at mouse mitochondrial DNA (mtDNA). In theory, a test for mouse mtDNA is all you need, because mtDNA is found in all mouse cells. In theory.

Now the four papers (or are they the Four Horsemen?) argue, in a nutshell, that mouse DNA shows up as "XMRV" on most of the popular tests that have been used in the past, that mouse contamination is very common - even some of the test kits are affected! - and that tests for mouse mtDNA are not good enough to detect the problem.
  • Hue et al say that "Taqman PCR primers previously described as XMRV-specific can amplify common murine ERV sequences from mouse suggesting that mouse DNA can contaminate patient samples and confound specific XMRV detection." They go on to show that some human samples previously reported as infected with XMRV, are actually infected with a hybrid of XMRV and a mouse ERV which we know can't infect humans.
  • Sato et al report that PCR testing kits from Invitrogen, a leading biotech company, are contaminated with mouse genes including an ERV almost identical to XMRV, and that this shows up as a false positive using commonly used PCR primers "specific to XMRV".
  • Oakes et al say that in 112 CFS patients and 36 healthy control, they detected "XMRV" in some samples but all of these samples were likely contaminated with mouse DNA because "all samples that tested positive for XMRV and/or MLV DNA were also positive for the highly abundant IAP long terminal repeat [found only in mice] and most were positive for murine mitochondrial cytochrome oxidase sequences [found only in mice]"
  • Robinson et al agree with Oakes et al: they found "XMRV" in some human samples, in this case prostate cancer cells, but they then found that all of the "infected" samples were contaminated with mouse DNA. They recommend that in future, samples should be tested for mouse genes such as the IAP long terminal repeat or cytochrome oxidase, and that researchers should not rely on tests for mouse mtDNA.
They're all open-access so everyone can take a peek. For another overview see this summary published alongside them in Retrovirology.

I lack the technical knowledge to evaluate these claims, no doubt plenty of people will be rushing to do that before long. (Update: The excellent virologyblog has a more technical discussion of these studies.) But there are a couple of things to bear in mind.

Firstly, these papers cast doubt on tests using PCR to detect XMRV DNA. However, they don't have anything to say about studies which have looked for antibodies against XMRV in human blood, at least not directly. There haven't been many of these, but the paper which started the whole story, Lombardi et al (2009), did look for, and found, anti-XMRV immunity, and also used various other methods to support the idea that XMRV is present in humans. So this isn't an "instant knock-out" of the XMRV theory, although it's certainly a serious blow.

Secondly, if the 'mouse theory' is true, it has serious implications for the idea that XMRV causes chronic fatigue syndrome and also for the older idea that it's linked to prostate cancer. But it still leaves a mystery: why were the samples from CFS or prostate cancer patients more likely to be contaminated with mouse DNA than the samples from healthy controls?

ResearchBlogging.orgRobert A Smith (2010). Contamination of clinical specimens with MLV-encoding nucleic acids: implications for XMRV and other candidate human retroviruses Retrovirology : 10.1186/1742-4690-7-112


petrossa said...

It's all explained in the Hitchhikers guide to the galaxy. Mice use us as testsubjects. ;)

Jon Brock said...

Fascinating stuff. So basically the CFS data are a lot noisier than we thought (and we can't trust them at the level of individual patients) but the retrovirus hypothesis still holds up. Unless of course there are systematic differences in the way samples are collected from patients vs controls which previously were thought unimportant.

Jonathan Hepburn said...

Mice are the vector for CFS, obviously. Which is why it's vital for all susceptible people to have cats.

Anonymous said...

Lombardi's paper did not show "immunity to XMRV", it only showed some low level of anti-XMRV antibodies. Nobody has shown that humans develop a significant titer, a high level of antibodies as expected from a viral infection. The macaques experimentally infected with XMRV made a nice high-titer response so we would expect humans to also.

Anonymous said...

@ Anonymous - Regarding the antibody levels - one key difference between the macaques and the CFS patients is that the macaques were tested for antibodies relatively soon after infection (within days and weeks) relative to the onset of illness in the CFS patients (tested within years to decades in presumed date of infection).

veri said...

HIV unlike most viruses doesn't occur naturally in the body. So what you're saying is this supposed contamination could have sinister applications as Petrossa hints we're not supposed to talk about. Wow. Are you sure you’re not a prophet? Years ago when I was invited to this bioterrism research launch I was expecting napalm, agent orange, but they rattled on about deformed mice, gene therapy or recombinant DNA technology. He said certain research was not for public consumption, even for politicians. The academia had a responsibility, he went all Che Guevara about it, it was moving but I thought he was nuts. Some scientists are already playing god and they don’t even realise it.

Neuroskeptic said...

Anonymous #1: OK, but the fact remains that Lombardi did show some degree of anti-XMRV activity which is hard to square with the idea that it's only mouse contamination; maybe the "anti-XMRV" antibodies in Lombardi were actually nothing to do with it and were just cross-reactive to XMRV but that's an open question afaik.

Neuroskeptic said...

Jon Brock: This study casts a lot of doubt on many of the previous reports of XMRV which relied only on PCR (namely, some of the cancer studies). And it also has implications for CFS although there are other lines of evidence there which don't rely on PCR.

Why the mouse contamination would be more common in patient samples than controls is unclear but there are plenty of ways it could happen. Maybe all the patient samples were recruited through a clinic which was contaminated, while all the healthy controls came through a blood donor program which wasn't, or something like that. We don't know, but it's quite possible.

petrossa said...

Lot's of things are possible but they tend to degrade when put against a likelihood axis.

The more you have to explain away loose ends the less likely a theory becomes.

And, as you suggest, most samples come from a contaminated source then the whole issue is null and void since the results are then invalid.

Anonymous said...

UK's Science Media Centre, with Simon Wessely on panel, responsible for science press releases for media to use

A reminder of the UK’s Science Media Centre, which, according to them, “is first and foremost a press office for science when science hits the headlines. We provide journalists with what they need in the form and time-frame they need it when science is in the news – whether this be accurate information, a scientist to interview or a feature article.” One member of their science advisory panel, whose “job is to to advise the SMC when their area of science hits the headlines, is Professor Simon Wessely Institute of Psychiatry, King’s College, London.

IMHO this could explain both why there has been basically no on-going coverage of all the XMRV–related research over the last year, and also why the pay-to-publish Retrovirology papers have been accompanied in the UK by press releases and a media blitz with headlines that go beyond the research and state that XMRV is a contaminate and/or that it is not related to ME/CFS.
– XMRV Global Action

Neuroskeptic said...

Some of the reporting on these new papers has been pretty crap. For example the BBC who said amongst other things that there's only one paper, from a British team - they don't mention the other 3 papers at all. They also quote the researchers as saying that XMRV doesn't cause CFS, but don't explain why they believe that, or why it's controversial.

But I don't think it takes Simon Wessely to cause bad reporting, bad science reporting is endemic in the UK at the best of times...

veri said...

I forgot to add, I guess the moral of the story is don't sign up as test subjects. If you really have a burning desire to do so, then make sure you do your research about the centre and make them sign a pre-nup. Your body, your life, you have a right to give them conditions too. Wow, that should so be a bill for parliament, which of course will lose because NGOs are poor, pharmas are not, and the judiciaries annul the universal declaration of human rights.. could the Vatican? hmm. Life is so hard.

JBME said...

"But I don't think it takes Simon Wessely to cause bad reporting, bad science reporting is endemic in the UK at the best of times..."

I agree, and believe the Science Media Centre takes a large share of the blame for that. There seems to be no such thing as serious investigative reporting on science or health issues in UK. The SMC is the lazy journalists' friend and we seem to have a lot of lazy journalists in UK.

I'd be interested in your opinion of what other diseases have had as consistently and persisitently biased and inaccurate media reporting over so many years as ME and CFS though.

The SMC's previous press release about the XMRV research was totally one-sided. No mention made of the Lo/Alter (NIH/FDA/Harvard)paper, which Dr. Alter says confirms the Lombardi et al (WPI/National Cancer Insitute/Cleveland Clinic)paper, finding MLVs, of which XMRV is a variant. The BBC made no mention of that paper either. UK reports always give the impression that the Lombardi study stands alone in finding XMRV.

This linked to the bias in funding by the MRC towards psychological "research" for ME/CFS, which Wessely, White and their colleagues benefit from while genuine ME patients are made worse by their "treatments", suffer and eventually die. It's a tightly woven web, spun for over 25years now.

As Dr. Alter said recently, the truth will out over the coming year.

veri said...

Covering a story on empirical findings leaves a lot of room for interpretation. Bad reporting is endemic all over the world. I reckon people don't realise because it usually isn't about them. There's way too much monopoly happening with the press. It isn't so free anymore. Saddens me.

Neuroskeptic said...

JBME: I think autism has been reported extremely badly in the British media, although it's got a lot better in recent years.

The vaccines-cause-autism meme, which has no place in a tabloid, was repeated very approvingly by most of the broadsheets and even Private Eye (otherwise an awesome magazine).

In that case, there was no conspiracy behind it, it was just journalists after a good story. Andrew Wakefield had no PR machine, if anything he quickly became an embarassment to the cause, but he didn't need one.

jace said...

On Monday five papers, not one, not four, were published consecutively in Retrovirology, a pay to publish journal, which showed only that the labs that produced the papers had contamination, and were unable to find the new human retrovirus, XMRV.

Dr. Jamie Deckoff-Jones, on her blog X Rx, said today "Nothing has been said of value in those five papers, except that future studies will have to be done carefully and that the authors are running sloppy labs."

The Cleveland Clinic, the National Institute of Health, Cornell University, the Whittemore Peterson Institute, the Federal Drugs Administration and the National Cancer Institute all have labs that can detect this family of Murine Leukemia related RetroViruses. Would such prestigious labs not take great care in their testing procedures?

The labs finding the virus, unlike the labs producing the papers published last Monday, used antibody, culture, and two other methods. The labs that say it is all contamination used PCR alone.

See the retraction of a similar article by Dr Vincent Raciniello, here:
23 December 2010 05:40:19 GMT

jace said...

The paper supposed to be behind the Wellcome Trust press release was Hue et al. The press release was definite that all XMRV was contamination, and while that is an exaggeration, all is not well with the source paper.
Quote "To consider the provenance of XMRV we sequenced XMRV from the cell line 22Rv1, which is infected with an MLV-X that is indistinguishable from patient derived XMRV."

The virus expressed by the 22Rv1 cell line  has been sequenced and has the unique nucleotide sequences in the env and gag regions which identify it as XMRV 123, therefore it is the same virus. The cell line originates from a patient suffering from prostate cancer in 1993. Taking the evidence as a whole the explanation that the origin of  XMRV expressed by these cells is that patient, in which case the difference in sequence was not derived from replication in an immortalised cell line but was representative of wild type XMRV in 1993 and yet Towers et al seem determined to adhere to their speculative viewpoint that this is a virus acquired by these cells, via passage through mouse cell lines. This seems irrational bearing in mind that there is no known MuLV virus which has the  characteristic 24 nt deletion in the gag leader or the same sequences in the env su region(1)

jace said...

I didn't mean to post that! It's part of a 25,000 character debunk of the Hue et al paper, with detailed referenced argument that pretty much destroys their reasoning. I realised it was not appropriate to this forum, but it posted anyway, as I navigated away :(

You can read the whole thing in the comments here

Neuroskeptic said...

Hey, no problem. All comments are welcome. Except Viagra spam...

jace said...

They;'ve moved the page I linked to in my last post. It is now here

Anonymous said...

Dear Dimwits,

All journals are "pay-to-publish", including Science, where the initial XMRV paper was published.

Neuroskeptic said...

Not all journals, some are free to publish in, however many respected journals, such as the PLoS ones, are pay-to-publish because they are free to access. Basically it's either the author or the reader who has to pay, unless it's funded by some company.

Anonymous said...

"Not all journals, some are free to publish"

Oh really? Care to list some examples for me?

Once again - all journals have publication fees. Open access journals have higher fees, because they are free for readers to access. Each journal has different policy and different rates. If you require color pictures/graphs, that will cost more per page than simply black&white. Most research grants include an amount of money set aside for publication costs. Some exceptions are made for research groups that for one reason or another cannot afford to pay, but overall it is extremely rare that the authors of an accepted paper do not pay fees of at least $1000 for publication.

Furthermore, even open access journals, which have higher fees are still peer-reviewed journals. Only after you have managed to get past the editors, and then are approved by the referees, will a paper will be accepted for publication.

This is just another example of how you XMRV brownshirts can't get basic facts straight.

the tainted cheese said...

"Not all journals, some are free to publish in"

Once again - examples please?

Or will you continue deleting replies because you know you're wrong.

Neuroskeptic said...

Sorry about your comment, it got caught in a spam filter, not deleted.

OK just for starters the British Journal of Psychiatry is free unless you choose to go for Open Access. The British Medical Journal currently charges a publication fee, but only under some circumstances, and until 6 months ago it didn't.

Anonymous said...

OK, well I guess any retrovirology study that is genuine should be published in the British Journal of Psychiatry. Is that the conclusion here?

For scientific journals, I think free publication is the exception rather than the rule, wouldn't you agree? Usually there are at least some costs.

Besides, the point is that paying higher fees for open access does not mean paying to avoid the peer review process, which was the implication of the comment I was responding to.

Neuroskeptic said...

Right, and I agree that implication is misleading. I was just pointing out that some journals are free to publish in, but no, they're not necessarily better because of that, far from it.

Anonymous said...

This is just another example of how you XMRV brownshirts can't get basic facts straight.

Is that you erv?

I find it particularly 'amusing' that anti-XMRV crowd think their appalling behaviour (see above) is a model of decency and scientific probity. Truth is that they are the ones who are behaving like brown shirts, while hiding behind a facade of 'concern for patient welfare' and 'getting the science right', blah blah blah.

Pure spin. Establishment thuggery.

Anonymous said...

Do you know what the image is of? It cannot be a contaminant.

Neuroskeptic said...

I'm not a virologist but my understanding of the latest argument is that XMRV is a real virus, but it's only present in mice, and (in the form of mouse DNA) in laboratory products.