XMRV UK CFS Study II: Dr. Vernon’s Analysis

February 16, 2010

Posted by Cort Johnson

As expected Dr. Vernon, the Scientific Director of the CFIDS Association, delivered a rather comprehensive overview of the latest XMRV study in the Retrovirology journal. Dr. Vernon spent some time making clear who just who did this study; it was basically the best of UK retroviral researchers (one ‘world-renowned’) plus top ME/CFS UK researchers with long histories of CFS research.

Like Dr. Shephard she clearly felt this paper presented a significant hurdle for XMRV. She reported that the PCR methods were identical to those used in the original paper. When those techniques didn’t find any virus they looked harder using a different, much more sensitive PCR technique. Dr. Vernon stated it could be ‘considered better and more sensitive’ than used in the original Science paper and they stil came up nothing.

If I understood it right, Dr. Vernon also put one limb of the Science paper’s argument in doubt by noting that the cross-reactivity in the antibody tests in this study suggested that the antibody tests in the Science paper could have been due to exposure to a different virus.

Again: the Wrong Patients? – The sample came from three cohorts. Dr. Vernon noted that because the blood from the biggest cohort came from relatively ‘new patients’ (1-4 years) it’s possible that XMRV doesn’t show up until later in the illness. This is not unheard of. My understanding is that HIV first attacks cells in the gastrointestinal system and that it can take several years before high levels of the virus occur in blood cells. The low copy level of XMRV suggests that it could have another locus in the body as well. Dr. Vernon discounted this idea to some extent by noting that Dr. Kerr, a prominent ME/CFS researcher (who derives his funding from several ME/CFS support groups), helped conceive the study and presumably would have taken care to include more ‘WPI-like’ patients in the other cohorts. Still it’s a possibility.

Did a longer duration, sicker, perhaps more immune suppressed group have a more detectable infection? Not according to Dr. Mikovits; she recently reported that patients in the study simply needed to meet the Fukuda or Canadian Criteria for inclusion; they were not particularly ill or disabled.

Still, Dr. Vernon took the WPI researchers to task for not releasing information regarding the illness duration, illness severity and treatment history of that original cohort, going so far as to suggest that their inability or unwillingness to do so could imperil the further research into the XMRV/CFS connection. She is clearly worried that more results like this one – which she warned will be forthcoming unless researchers know which patients to study – will dampen scientific interest in XMRV and CFS.

Hiding out in the UK but not the US? – She also noted that the virus could be present in such low levels that even with their more sensitive techniques the Retrovirology researchers couldn’t find it. That begs the question, though, why the WPI with their less sensitive methods was able to find it both before and after the Science paper in both US and UK patients. (Dr. Mikovits noted that VIP Dx labs sometimes searched 3 or 4 times before they found the virus – did the WPI researchers do the same with their Science samples?)

The virus is clearly hard to find, that’s for sure; of the 27 people on the Phoenix Rising Forums VIPDx Poll only about 20% tested positive to the PCR test but 20% is still light years different from the zero percent the Retrovirology researchers reported; given their background they clearly would have loved to find 20% positive rates.

Yes, perhaps there weren’t enough long duration and severely ill patients in the Retrovirology study and the Imperial College study but it still seems hard to believe that the wrong patient cohort is the cause of “zero percent” positive rates. The cause of the conflicting study results is still very unclear.

Dr. Vernon made it clear that the DHHS studies will use different techniques, including the WPI’s original techniques, to examine people who tested positive for XMRV via the VIP Dx lab test. This will help determine why the discrepancies are showing up.

Everyone’s Doing Everything Right!
- This has been a tough couple of days for XMRV but check out this take from a researcher in ScienceNow. It suggests that disparate results happen even in the best labs.

“discrepancies between labs are common, says David Griffiths, a virologist at Moredun Research Institute in Midlothian, United Kingdom, who has studied previous claims for retroviruses as the cause of chronic diseases. He also notes that he cannot find serious flaws with any of the published studies: “All the people involved are doing things exactly as they should be.” For the time being, then, the XMRV results will remain frustratingly ambiguous. As Griffiths says, “There must be an explanation for why disparate results are showing up, but it may not be an easy thing to turn up.”

For the Full Text of Dr Vernon’s Analysis

15 comments

{ 15 comments… read them below or add one }

John February 17, 2010 at 9:14 am

One other thing I’ve read is that the WPI will culture their samples for 4-5 days before doing the PCR in an attempt to increase the amount of XMRV present in the sample to be tested.

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admin February 17, 2010 at 4:16 pm

Thanks for providing these insights John. This and the idea that the WPI is taking multiple passes through the samples to find the virus do appear to provide legitimate reasons for the discrepancies (at a time where there don’t seem to be too many other answers). It’ll be interesting to see if the WPI provides them in their response to the study.

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Steve Fenney February 17, 2010 at 3:51 pm

Everyone’s Doing Everything Right!….???????????????????

Sorry, I can’t see it that way. Sure, I understand that you need independent studies to corroborate any conclusions, but if you’ve got a theory which states that XMRV is implicated in ME (not CFS!), then you need to get heads together to work it through optimally. This process is just wasting time and energy.

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admin February 17, 2010 at 4:12 pm

I think you’re right to some extent and it is happening with the DHHS group – they appear to be using the exact methods as the WPI – and are using other methods to figure out where the discrepancies have occurred.

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John February 18, 2010 at 12:11 pm

Maybe the WPI does or doesn’t look multiple times at samples, including in their Science paper- there are so many comments made on specific examples which are then possibly incorrectly generalized(I try not to, I swear!) that I for one can’t tell what’s what.

From the WPI’s response,
-” Perhaps the most important issue to focus on is the low level of XMRV in the blood. XMRV is present in such a small percentage of white blood cells that it is highly unlikely that either UK study’s PCR method could detect it using the methods described. Careful reading of the Science paper shows that increasing the amount of the virus by growing the white blood cells is usually required rather than using white blood cells directly purified from the body. When using PCR alone, the Science authors found that four samples needed to be taken at different times from the same patient in order for XMRV to be detected by PCR in freshly isolated white blood cells. More importantly, detection methods other than PCR showed that patients whose blood lacks sufficient amount of XMRV detectable by PCR are actually infected. This was proven by the isolation of viral proteins and the finding of infectious XMRV isolated from the indicator cell line LNCaP. The authors of the Retrovirology paper admit that their neutralization assay did not detect bacterially expressed XMRV gag and that positive control sera was needed to validate their assay… A failure to detect XMRV is not the same as absence of this virus in patients with CFS.”

***********************
WPI’s response to second negative UK/XMRV study-
http://www.facebook.com/notes.php?id=154801179671

February 18, 2010: WPI is aware of the recent UK study that was unable to detect the presence of XMRV in any CFS patient samples. Although researchers at the WPI were not involved in this project, our work in XMRV continues with researchers around the world. We look forward to the results of studies which replicate the methods used in the original research described in the journal Science in October, 2009.
View more…

Information Regarding XMRV Studies

1. The authors of the Science paper established the existence of XMRV as an infectious human blood borne retrovirus for the first time in blood of patients diagnosed with Chronic Fatigue Syndrome (CFS). Previous studies had established the presence of XMRV sequences and protein in human prostate tissue.

2. In the Science paper, the presence of XMRV in well-characterized patients with CFS was established 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.

3. The authors of the two UK studies did not attempt to “replicate” the WPI study. Replication requires that the same technologies be employed. The WPI sent reagents and information to several groups of researchers in an effort to support their replication studies. Neither UK study requested positive control blood, plasma or nucleic acids from the WPI.

4. The collection, preparation and storage of DNA were completely different between the Science and UK papers. The latter studies do not show data on blood harvesting or storage. Nor do the studies disclose the quantity of isolated cells. Insufficient number of cells analyzed may result in failure to detect a low copy virus like XMRV, regardless of the sensitivity of the assay. Neither UK study provides detail to allow interpretation of how many white blood cells were analyzed.

5. Patient population selection may differ between studies.

6. The UK authors were unable to detect XMRV, even though 4% of healthy individuals were found to be infected in the US. Japanese scientists detected XMRV in 1.7% in healthy blood donors in Japan. The two previously identified human retroviruses have distinct geographical distributions.

7. Perhaps the most important issue to focus on is the low level of XMRV in the blood. XMRV is present in such a small percentage of white blood cells that it is highly unlikely that either UK study’s PCR method could detect it using the methods described. Careful reading of the Science paper shows that increasing the amount of the virus by growing the white blood cells is usually required rather than using white blood cells directly purified from the body. When using PCR alone, the Science authors found that four samples needed to be taken at different times from the same patient in order for XMRV to be detected by PCR in freshly isolated white blood cells. More importantly, detection methods other than PCR showed that patients whose blood lacks sufficient amount of XMRV detectable by PCR are actually infected. This was proven by the isolation of viral proteins and the finding of infectious XMRV isolated from the indicator cell line LNCaP. The authors of the Retrovirology paper admit 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.

Simply stated the only validated reliable methods for detecting XMRV in CFS patients, to date, are the methods described in Science. Failure to use these methods and validated reagents has resulted in the failure to detect XMRV. A failure to detect XMRV is not the same as absence of this virus in patients with CFS.

source
http://www.wpinstitute.org/news/news_current.html

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mherschen February 19, 2010 at 6:40 am

We are being led on a wild goose chase with XMRV. Even if XMRV is implicated, it’s not the virus itself that “causes” CFS, it’s the disregulated immune system. My CFS was triggered in one day after having an inflammatory immune reaction to a supplement, including fever, swollen glands, aches and pains, chills, etc. Did I not have XMRV before and now, with no exposure to anything (I was alone in my house for days/weeks before and after) suddenly contract XMRV out the air? Of course not – it’s ludicrous.

Even if the XMRV were there latent, ready to activate upon the immune disregulation, the fact would therefore be that I can have XMRV without having CFS. The immune system is the problem, not the retrovirus. You can replace “XMRV” with anything you want: EBV, HHV6, Lyme (all of which I also have), and probably a hundred other things, some of which some people have, some of which other people don’t. That means it’s not the virus or pathogen in itself that’s important in causing the disorder, it’s the state of the immunity that’s causing it, and which needs to be fixed.

Finding XMRV is not going to fix the immunity. At best — even if the virus IS sometimes implicated — finding it is only going to give an avenue for *maybe* treating with anti-retrovirals, most of which undoubtedly will be relatively toxic even to healthy people. Because that is true, the mainstream doctors are not going to afford you, me, us with any of the so-called “validation” so many people seem to care about and want. That’s because the mainstream doctors/scientists will only admit something is real that they know how to fix or at least partially treat. They don’t know how to repair the immune system – the don’t know virtually anything about the immune system, actually, meaning anything functional and the way it interacts with the neurology, with the psychology, with the society (i.e., psychosocial neuroimmunology). So we are going to be as marginalized as ever, still having to search for “alternative” doctors to give out anti-retrovirals (if the FDA or some similar agency) doesn’t shut them down and make that impossible, too.

I know I be will attacked for providing my opinion because people want to believe the XMRV research will provide some miracle treatment, but I think they WPI people are barking up the wrong tree. To fix or even cure CFS you have to fix the immune system. WPI wants/needs to make money just like anyone else. I’m not impugning their findings, only saying don’t put all your eggs in someone else’s one basket. Right now the best hope for that appears to be stem cells, not in questionably toxic anti-retrovirals for some mysterious retrovirus that some people have, others don’t, and that doesn’t to cause CFS on its own even if the person does have it. Put your time, money and energy into stem cell advancements, and lowering the cost and increasing the availability of stem cell therapy. That’s where we will get our cure.

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Gail (Upnorth) February 19, 2010 at 11:01 am

Thanks for your commentary and updates on the XMRV research Cort. I’ve been glued to your “buzz” page and check it every day. When this second UK study came out and I saw the names on it (Kerr etc.) my heart dropped into my gut I was so disappointed. I’ve always suspected deep down that for many of us, there is some kindof viral root to this illness. The Science paper in the fall was enough to get me actually hopeful. Now, like you mentioned, it looks to be a very bumpy road for XMRV. DO you know of any papers in the works (outside of Whitmore/Peterson) that have found XMRV in patients?…Vernon mentioned in her talk yesterday that several of the CFIDS funded projects are now also testing for it..

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admin February 19, 2010 at 12:03 pm

I don’t think the road for XMRV is nearly over. The WPI posted a very interesting response to the latest study – which, at least to my laymen’s mind – contained some good reasons why the search for XMRV thus far has been unsuccessful. On the XMRV studies page there are lots of groups that are looking for this which suggests to me that we will get to the bottom of the XMRV question http://aboutmecfs.org/Rsrch/XMRVStudies.aspx

(I’d better got back on the Buzz page!)

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Laurence aka Wingfingers February 19, 2010 at 5:06 pm

Today (19th Feb) is the 15th Anniversary of my coming down with a “flu-like” illness which matured into M.E. My GP didn’t give me a diagnosis but he did give me painkillers and antidepressants – fat lot of good they did. To me, I had caught a flu-like virus or something similar, but it took me 3 months with a serious headache before I even visited the doctor. As a veterinarian, I normally treat myself for minor ailments, I know as much as the GP knows and he knows it.
I do not stand tall, but I expect people discussing serious diseases to keep a fully open mind. I personally am pretty certain that XMRV is the real cause of M.E. – it’s so obvious. It depresses the immune system, just like its close cousin HIV, so you do get rises in levels of HHV6, enteroviruses, glandular fever, all the usual viruses that get implicated even if they are secondary. And like HIV, it can be transmitted via body fluids. I’m deliberately not using the word s3xual; I think it’s worse than that. It can certainly be transmitted by airborne methods, unlike HIV. See “Osler’s Web” by Hillary Johnson. I had an affair with an old girlfriend – we didn’t have a s3xual relationship in those heady teenage years – but she tried to make a comeback, with both of us having married, had 2 kids each, and felt let down by our spouses. She’d been the light of my life back in those old days, I never forgot her. Significantly, she’d had a period of depression (2 or 3 years) following the death of her father when she was about 15. She was now a CPN (community psychiatric nurse) so did not “believe in” M.E. On Valentine’s Day 1995 she relented her rule about “only straight sex” and we did exchange body fluids. 5 days later, I came down with M.E. I am certain that the WPI research will be vindicated in due course, but I just wish these deniers would stop putting a negative spin on everything – they will be wrong in the end.

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mherschen February 20, 2010 at 6:47 am

@Laurence aka Wingfingers:

No one’s putting a “negative spin” on anything. It’s you who are putting an unscientific and unreasonable pollyana “positive spin” on things. Your thinking is flawed in many ways, like for example when you say you are “pretty certain” and that it’s “so obvious” CFS/ME is from XMRV, when there is no real evidence for that at all.

You also make a post hoc ergo propter hoc fallacy by concluding that having sex with your girlfriend gave you the retrovirus. You also make many over-the-top “conclusions” and assumptions about airborne and other routes of transmission. Again, none of that has been proved in the least.

You also imply through faulty reasoning that an antioxidant berry supplement must have contained XMRV and that it’s therefore possibly to contract XMRV through ingesting food substances contaminated with XMRV, because I was not sick with CFS before taking the berry supplement that caused the immune activation. Since you claim it is “pretty certain” that XMRV causes CFS, I could not have had XMRV before taking the berry supplement, because I did not have CFS then. I only had it the next day. So where did the XMRV come from if all other variables were the same?

So it’s really you who are not open minded. You have made up your mind without any real or compelling evidence whatsoever that XMRV “causes” CFS, and you apparently have closed your mind to everything else. Naturally you are going to be upset when you hold such an unrealistic, unscientific view and someone comes along who is a little more grounded in reality and points out the facts, which are that immune system dysregulation appears to be at the core of CFS/ME, not any particular virus, and that the immune system has been witnessed to return to a normal state in 100 percent of CFS stem cell recipients. It’s also a fact that a few cures have been attributed to stem cells, which obviously did not include any anti-retrovirals in the infusion, only stem cells.

So if you are really to be open minded, you would need to engage those facts, as well as the fact that WPI has an agenda just as any other group has. That doesn’t mean they’re wrong, but it also doesn’t mean they are automatically right, just because you share the same agenda. That is unscientific. We need to go where the facts lead, not where we want them to lead.

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Amaia February 25, 2010 at 10:57 am

I agree with you Mherschen………….inmune system is the key..not the XMRV…

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alphahusky March 2, 2010 at 9:00 am

mherschen-

I don’t like to argue with people about the cause of their disease, as many people attribute it to many other things. The berry supplement issue- perhaps you did have some issue with your immune system and this caused the crash, led to severe illness, etc. But as much as you are arguing with those of us who *did* have a very clear viral start to this illness, I have to say something that popped into my head immediately, “Correlation does not imply causation.”

You could say this to me as well. You could say, well, your severe case of Mono that happened to be identical to the disease you carry now, 25+ years later, minus some symptoms and plus some more- same thing! Perhaps a coincidence. It could be- I had some*thing* going on with my immune system after an “experimental vaccine” a few years earlier. To me, that was what introduced the potential for the disease. For me, I know perfectly well that day before Mono, healthy and strong. 3 months later, 6 months later, 2 years later, 25 years later- eh, not so much. Not even at all. For me, the extreme similarity between Infectious Mono (aka Glandular Fever) and what I have to this day means there had to be some kind of viral trigger. The fact that I pretty much had every symptom of AIDS when it was first talked about on the radio in early 1986 has convinced me through all these years that a virus plays the biggest part in this disease.

I know longer know what virus. I, too, held out lots of hope on the various viral theories over the year, though of course the start of this disease for me was far clearer, though it sure didn’t help anyone believe it was real. I don’t know if XMRV started it, if it just piggy-tailed onto something else, or if it happens to simply be in some people who also happen to have this disease and it plays no part. I would have sworn to you up and down that Epstein-Barr had to have some causative role in this. When I recalled that I’d had that vaccine and shortly after began to show immune system regularities and got a bunch of strange and more severe-than-normal illnesses (though nothing what I’ve lived with for a quarter century), I had to start wondering if the initial trigger or cause was something about or in the vaccine.

I do know one thing. Everyone piling onto the backs of those of us who had some apparent viral trigger to the disease itself isn’t helpful either. Whether or not your disease was *caused* by a berry supplement isn’t something I’m qualified to say. But just because something happens on the same day as something else doesn’t necessarily mean it caused it, or else Epstein-Barr would have been an absolute, unchallenged cause to this disease in me, at least. There is always the possibility that you had some virus in your system that a berry supplement, an antioxidant, permitted to circulate more freely in you, causing the crash. But as many would like to tell us all that there is no virus proved to *cause* ME/CFIDS; there is also no supplement proved to cause it.

I don’t trust the US government or the UK government much either, or their reps in this field, not because of the lack of evidence of this or that cause, but because they insist that it’s made up, a psych coping strategy- anything but a physical disease. They made us into a laughing stock, so whether this is brought on by an inherited immune system problem, a virus, a supplement, a combination, whatever, all we are is a joke to many of them. In this day and age, I can’t imagine any other disease- whether inherited or transmitted- that is the subject of so many jokes and disparagement, let alone by scientists. Not implying that is happening here, of course, but it is something that always irks me. I just know that because it appears to be so easy to say “this is a mental illness,” I am always wary of people with the disease who spend time arguing with each other over the cause. As you say, the day before the supplement, you didn’t have this. The day after, you did. It’s a reasonable conclusion. Viruses take longer than one day to incubate; they sometimes take much longer to spread or show effects. For me personally, that makes more sense as a cause or trigger or whatever you want to call it. I’d much rather have a bunch of scientists going out of their way to prove or disprove whatever than joking about all of us.

But our lived experience as victims of this tragic illness- I’m not going to start saying, “Evidence, please!” when we all know perfectly well that our disease is underfunded in studies and has rarely been taken seriously by those who should have been paying attention in the beginning. Honestly, or else you’d have to *prove* a supplement caused this, or we’d all have to have sensitive immune system tests from birth to prove it was some defect in our immune systems.

Well, I’ve lost concentration due to my child insisting she needs a diaper change. Now that, I can easily prove!

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John March 22, 2010 at 3:02 pm

Cort, I’ve seen you repeat several times now the part about the WPI looking multiple times, and can’t figure out what your exact views on it are. From #7 in the WPI’s response- “Careful reading of the Science paper shows that increasing the amount of the virus by growing the white blood cells is usually required rather than using white blood cells directly purified from the body. When using PCR alone, the Science authors found that four samples needed to be taken at different times from the same patient in order for XMRV to be detected by PCR in freshly isolated white blood cells.”
http://www.facebook.com/notes.php?id=154801179671

What this says is that it is necessary to culture the sample in order to increase the amount of virus present in order for it to be detected by PCR. It is only when culture is not used, ie ‘PCR alone’, that the WPI needed to look multiple times to find XMRV by PCR. They didn’t look multiple times at multiple samples in the Science paper, because it wasn’t necessary as they cultured all samples. It was only when they tried to find the virus ‘by PCR alone’, ie without culturing it, that they needed to look at multiple samples, at least from how I’m reading their reply.

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admin April 11, 2010 at 4:05 pm

Thanks for clearing that up John.

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John March 22, 2010 at 3:06 pm

Whoops, this is the correct link to the WPI’s response- http://www.facebook.com/note.php?note_id=311221958025

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