Deja Vu in the UK – XMRV CFS Study Negative Again!

February 15, 2010

Posted by Cort Johnson

UK researchers are not winning the hearts and minds of CFS patients – that’s for sure. Just a couple of uplifting weeks after Dr. Mikovits displayed so much enthusiasm and confidence in XMRV the other shoe has dropped. An Imperial College researcher said another negative study was coming and here it is; this UK study also failed to find virtually ANY evidence of XMRV in a large number of CFS patients. This study was similar and different from the Imperial College study.

Annette Whittemore said to be cognizant of who’s doing the studies – in this case, though, there doesn’t appear to be any bias to question, no damning history of behavioral emphasis to reflect upon; two members of the study, Dr. Kerr and Dr. Gow, are long term ME/CFS researchers committed to a pathophysiological interpretation of this illness. (Ironically it was Dr. Gow that refuted Dr. DeFreitas finding 25 years ago).

Indeed, the paper went to some lengths to praise the Lombardi Science paper stating the “apparently compelling evidence against the possibility of laboratory contamination” and the immune response against XMRV the researchers demonstrated was present. They stated that they set out with ‘the intention of confirming the Lombardi’ study.

PCR Tests - This was a large study that looked at well over 500 CFS patients and controls from two cohorts in the UK and Scotland. They first looked for sequences on two the three genes XMRV possesses. When they didn’t find anything the first time they looked again using a more sensitive assay.

Immune Tests
- Unable to find evidence of XMRV by PCR they looked for signs that the patients immune systems were reacting to it. To do this they obtained some ‘neutralizing antibodies’ against the ‘env’ protein found in the family of mouse retroviruses. Antibodies neutralize retroviruses by attaching to them and preventing them from getting their hooks into cells. They also raise a red flag to the immune system to come and attack. As they examined this set of antibodies they were able to identify one that was specific for XMRV and they used it to search for the virus.

The neutralization test is a rather indirect one; they apparently add the antibodies to the sample and then (somehow) test the sample for ‘infectivity’. Since the antibodies attach themselves to the retroviruses the degree of infectivity should go down a certain amount and in a couple to test cases they confirmed this. When they ran the neutralizing antibody test on the 142 ME/CFS patients none of them met the criteria for infection. Ironically, 14% of the healthy controls from one of the healthy cohorts tested positive for infection, altho later testing suggested it was do to a different mouse virus.

They stated that they were ‘confident’ that their ‘PCR assay is more sensitive than the published single round PCR method and should have possessed the necessary sensitivity to find XMRV’.

Two Different Tests : Two Different Results – The WPI has backed away from the PCR test because of its inability to detect XMRV at very low levels and their associated lab VIP Dx is not longer offering it. This could not be a reason, of course, for the zero results seen in this test – the WPI’s PCR test may not be perfect but it appears to be able to find most instances of infection. We also know from Dr. Lombardi and from patient reports that the WPI’s test IS finding XMRV infection in UK patients. Why they are finding it and two UK groups have not, is, of course, the big question. Either the patients are very different or the tests are. Since it seems unlikely that that the patients are THAT different its pretty clear that the WPI’s test is quite different from these other groups.

Validation Not a Replication Study – It’s interesting, by the way, that this UK group – with its ties to the WPI via Dr. Kerr – did not appear to avail itself of the WPI’s assays or or Dr. Singh’s antibody tests. Since the group didn’t appear to use the WPI’s methods this is a validation study not a replication study; its was an attempt to validate the WPI’s claim that they’d found XMRV not an attempt to determine if the the WPI’s methods worked.

This is still just the beginning of the XMRV studies to come out but its a remarkable turnaround given the lengths the WPI, researchers from the NCI and the Cleveland Clinic went to in that compelling Science paper (Dr. Coffin called it as good a first paper as they get) to demonstrate the presence of XMRV. The fact they were able to show that this virus was able to infect previously uninfected cells and show a virus budding out of them still seems – at least to this layman – to be the most singular and important finding to date.

My understanding is that the WPI is now using Dr. Singh’s antibody test specific to XMRV that shows INCREASED not decreased rates of positivity. It really is a conundrum; the WPI appears to be getting more and more internal evidence that they’re right these papers are coming out suggesting that something went wrong. The first question always appears to be if what the WPI found is an endogenous retrovirus – a piece of junk DNA from an old mouse retrovirus in our genome. They sequenced 2 and a half strains of the virus and compared what they found against our entire genome against our entire genome and found nothing. That’s one of the reasons Science took the paper – they convinced them it was not an endogenous retrovirus. But if it’s not that then what is it?

The Scientific Director of the CFIDS Association, Dr. Vernon, will reportedly release an analysis of the study tomorrow, giving us a much needed expert overview of the situation.

{ 12 comments… read them below or add one }

Charley February 16, 2010 at 5:48 am

Sadly than LNCap image of the budding retrovirus isn’t good evidence – it could be any virus, and you should be able to tell from an electronmicrograph whether it’s a retrovirus or not (they look distinctive). This one… could be anything :(

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admin February 16, 2010 at 7:11 am

Thanks, so all we know – based on the photograph – is that some virus jumped from the CFS patients cell to the clean cell.

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admin February 16, 2010 at 7:48 am

My understanding is that the WPI is now using Dr. Singh’s antibody test specific to XMRV that shows INCREASED not decreased rates of positivity. It really is a conundrum; the WPI appears to be getting more and more internal evidence that they’re right these papers are coming out suggesting that something went wrong. The first question always appears to be if what the WPI found is an endogenous retrovirus – a piece of junk DNA from an old mouse retrovirus in our genome. They sequenced 2 and a half strains of the virus and compared what they found against our entire genome against our entire genome and found nothing. Thats one of the reasons Science took the paper – they convinced them it was not an endogenous retrovirus.

Bear in mind that we have yet to see a ‘replication study’; no one has yet followed the WPI’s study to the letter. Different groups are doing different kinds of PCR and different kinds of antibody tests. Theoretically they all should match up but they’re not; the twists to this story are amazing and unsettling but the WPI has the National Cancer Institute and the Cleveland Clinic behind them; they produced ‘the best’ first paper possible and it landed in the most prestigous journal in the world. Other groups are doing more comprehensive analyses of the WPI results.

I still don’t see how they could be wrong. I think the WPI has still done more to show that the virus is real than anyone has shown that its not. How could they be wrong? (How could they be right?) ; Dr. Klimas said this was going to be an up and down process – she was clearly right! The twists in this story are amazing.

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John February 16, 2010 at 8:29 am

One criticism I’ve heard of the WPI’s techniques states that the WPI uses a methylation inhibitor before testing cells.

The criticism is that the methylation inhibitor stops the cells from doing what they do naturally, ie inhibiting ERV’s. But I don’t see how a ‘reactivated’ ERV would match up so well on a phylogenic tree as the WPI’s XMRV does.

Plus one other thing I just thought of- Dr. Mikovits has stated that the WPI tried several times to find patient positives. Did they go through the same rigour trying to find healthy positives? I don’t know if they could have because I don’t know if they have longitudinal samples from the controls, but that seems like it really could have made a difference. If you look through the patients’ samples two or three times to get a positive and don’t do that for controls as well, that might skew the ratio of positives to negatives.

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John February 16, 2010 at 8:35 am

Plus you have the cancer aspect, both historically as reported in Osler’s Web (weren’t some patients collected by Dr’s Peterson and Cheney actually excluded by CDC prior to the creation of ‘Chronic Fatigue Syndrome’ because they had cancer?), plus the cohort Dr. P is following right now and has been for many years(which I think the NCI is studying now as well) with mantle cell lymphoma. That fits in hand in glove with a virus known to cause neurological disease and cancer in mammals, doesn’t it?

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richard February 16, 2010 at 2:43 pm

Dr. Kerr did not collaborate with WPI on this

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Renee February 17, 2010 at 11:28 pm

Did the UK group use the same criteria to select patients as the WPI? Maybe there are 2 different criteria in use, giving rise to the disparate results.

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admin February 18, 2010 at 7:55 am

They used similar criteria; the WPI required that patients fulfill either the Fukuda OR the Canadian Consensus Criteria – the latest study required that they fulfill the Fukuda criteria. Since many patients fulfill both that can’t be the cause of the zero results. Dr. Vernon is wondering – and has obviously worried from the beginning – if duration of illness or severity plays a role. She believes those criteria may be very important.

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Mary Schweitzer February 19, 2010 at 5:29 pm

You’ve missed the main point. What we need at this stage is REPLICATION studies. That means following the WPI protocol precisely, whatever you think of it. I was immensely disappointed that Kerr chose to do his own thing (for whatever reason), leaving the false impression the study showed there was something wrong with the WPI/NCI/Cleveland Clinic results. (Note that they took great pains in an accompanying note to say it would be improper to conclude they had somehow disproved WPI/NCI/Cleveland.

WPI offered to share; they refused.

Whatever the reason behind the refusal of Kerr to follow the procedures for replication, we are left no wiser than before.
“Science” really put the WPI and partners through a lot of tests – they already have gone through steps to show there was not contamination in the lab, for example.

I don’t think it takes deep thought to understand why McClure-Wessely would have botched the job – but I remain perplexed as to why Kerr-Gow did not follow the study procedure. The only answer I can come up with is that they hoped they would get positive results using a different (and marketable) method.

Mary Schweitzer

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admin February 19, 2010 at 7:21 pm

It’s true that we need replication studies but that was not a given 3 weeks ago. The reason XMRV has not shown up in these studies is still obscure. Kerr et al had every reason to expect they would find XMRV; they had markers for it that they believed should have done the job- they used very sensitive – Dr. Vernon stated they were probably the most sensitive – tests, they looked twice using two different tests and they didn’t find it. They weren’t trying to invalidate the WPI’s findings- they were trying to validate them. It’s very clear now, though, that replication studies ARE needed and they appear to be forthcoming.

If they had validated the WPI’s results we would be in BETTER shape than if they’d simply tried to replicate because you would have had independent verification of the WPI’s finding using different techniques; XMRV would have been rock solid in the research world at that point. Unfortunately that didn’t happen.

We are left wiser (if not happier :)) than before; we know that standard techniques even with very sensitive tests will not find this virus – you apparently have to be very careful in how you look for it.

As the doctor at the end of the blog stated “Everyone’s done everything right” – this appears to be more of a case of an unusual bug than anything else.

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admin February 20, 2010 at 7:45 am

I will say, though, that after looking at the methods it’s really unclear to me – a laymen, for sure – why the Groom study didn’t culture their cells. I believe there’s only one reason to culture cells and that’s to up level of a low level virus. The Science paper has three sections where they culture the cells; they obviously did that for a reason – yet the Groom group ignored that. It seems inexplicable to me that they could have ignored such a fundamental aspect of the study. Perhaps they thought their increased sensitivity would have made up for that.

Someone on the Phoenix Rising Forums just posted a list of standard blood isolation and amplification procedures for HIV http://forums.aboutmecfs.org/showthread.php?3133-XMRV-CFS-UK-study-II/page52 and stated that the WPI followed most of them and the UK groups followed few of them. If you look at the methods sections of both papers you’ll see that the methods section for the relevant procedures are longer in the WPI paper than in the UK papers.

Maybe it is a case of poor technology for UK researchers; they are not known for their cutting edge techniques.

John February 20, 2010 at 10:47 am

To make an update to my earlier post- “One criticism I’ve heard of the WPI’s techniques states that the WPI uses a methylation inhibitor before testing cells.

The criticism is that the methylation inhibitor stops the cells from doing what they do naturally, ie inhibiting ERV’s. But I don’t see how a ‘reactivated’ ERV would match up so well on a phylogenic tree as the WPI’s XMRV does.”

From looking at the pdf of Dr. Mikovits’ presentation at that hotel, I think at the end it says that the methylation inhibitor was used in the samples that did not test positive in the Science paper, and not in the original samples. I was wondering how that would have escaped unnoticed if they indeed had used it in the Science paper!
http://www.wpinstitute.org/news/docs/WPI_JAM_012210.pdf

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