XMRV CFS UK study #II

[julius]

We still dont know whether those samples contained XMRV or not but they concluded that the results were negative if this isn,t a fatal flaw i dont know what is.

Yes, the conclusion was also incorrect. And a good example of bad science.

 

[Mithriel]

Science does not include details of how the tests were done in original papers, these are available as supplementary information. My virologist friends feel that the details given in these by the WPI were very good and better than those seen in a lot of papers.

The reason for multiple samples is that XMRV does not replicate very often. It is only at those times the virus is replicating that it is free in the blood and able to be found by PCR. This means that only a sample taken at the right time will give a positive result unless you stimulate the cells in some way that encourages the virus to begin replication. Some patients needed blood taken at four separate times before the researchers caught the virus in the act of replicating.

When patients have been sending in blood to be tested XMRV is not being found in their blood unless it is actively replicating. If they culture the blood the virus gets coaxed into replicating so you get negative by PCR positive by culture.

Now they understand this they have stopped testing blood by PCR.

The WPI studies were done in more than one lab so they have already been replicated, that's why Mikovitz is confident that external replication will work if they follow the correct technique. This was a good, well done study, well planned and well executed.

The Kerr study was done quickly using things they already had to hand. They did not follow the WPI procedure because they did not want to do so. It wold have taken money and time to set up. There was no small deviation caused by a step left out of the Science paper, they made no attempt to follow the same procedure. This was not necessarily a bad thing, it might have worked but it was a decision they made, not something caused by sloppiness on the part of the WPI

They chose to use banked blood, probably also because of time and money constraints. Again it might have paid off but it didn't.

As for the Imp C study, school projects take longer to set up. This was rushed in the hope of putting a stop to research and the conclusions they reached were laughable if they were not so dangerous to us.

I stand by what I said. The Kerr study should have used a positive specimen of blood to test their procedure and only tested patients, banked blood or fresh, once they had perfected their technique. The fact that they didn't was a serious flaw.

I do not see why stating this means I am holding it up to a higher standard than the Mikovitz study. I am looking at both studies with a clear eye, I have a scientific background and virologists agree with me. It's time to let the WPI get on with their work for us.

Mithriel

 

[ukxmrv]

Just as an item which may be of interest to some. Dr Gow was involved in the 2nd attempt. I found an earlier criticism of his attempt at the DeFreitas virus

-----------------------------------------------------------------------------------

Another of these five reports is that of Gow el al from laboratories in
Scotland and Germany (5). Again using the original DeFreitas et al.
primers, as well as another HTLV-2 primer pair, no differences were
observed between CFS patients and control subjects (100% of both groups of
subjects were positive for HTLV-2 gag gene related DNA sequences).

Weaknesses of this study include:

• the use of a European case definition for CFS (6) which differed
substantially from that used in the United States

• failure to use a "hot start" PCR procedure such as that used by DeFreitas
et al.

• use of the DeFreitas et al. primers with reaction conditions
significantly different from those used by DeFreitas et al

• the DeFreitas et al. detection DNA probe failed to recognize the PCR
product obtained, suggesting nonspecificity of the PCR reaction.

 

[Gerwyn]

Science does not include details of how the tests were done in original papers, these are available as supplementary information. My virologist friends feel that the details given in these by the WPI were very good and better than those seen in a lot of papers.

The reason for multiple samples is that XMRV does not replicate very often. It is only at those times the virus is replicating that it is free in the blood and able to be found by PCR. This means that only a sample taken at the right time will give a positive result unless you stimulate the cells in some way that encourages the virus to begin replication. Some patients needed blood taken at four separate times before the researchers caught the virus in the act of replicating.

When patients have been sending in blood to be tested XMRV is not being found in their blood unless it is actively replicating. If they culture the blood the virus gets coaxed into replicating so you get negative by PCR positive by culture.

Now they understand this they have stopped testing blood by PCR.

The WPI studies were done in more than one lab so they have already been replicated, that's why Mikovitz is confident that external replication will work if they follow the correct technique. This was a good, well done study, well planned and well executed.

The Kerr study was done quickly using things they already had to hand. They did not follow the WPI procedure because they did not want to do so. It wold have taken money and time to set up. There was no small deviation caused by a step left out of the Science paper, they made no attempt to follow the same procedure. This was not necessarily a bad thing, it might have worked but it was a decision they made, not something caused by sloppiness on the part of the WPI

They chose to use banked blood, probably also because of time and money constraints. Again it might have paid off but it didn't.

As for the Imp C study, school projects take longer to set up. This was rushed in the hope of putting a stop to research and the conclusions they reached were laughable if they were not so dangerous to us.

I stand by what I said. The Kerr study should have used a positive specimen of blood to test their procedure and only tested patients, banked blood or fresh, once they had perfected their technique. The fact that they didn't was a serious flaw.

I do not see why stating this means I am holding it up to a higher standard than the Mikovitz study. I am looking at both studies with a clear eye, I have a scientific background and virologists agree with me. It's time to let the WPI get on with their work for us.

Mithriel

I agree with every word.I have a science backroud in this area and i have reached the same conclusions my eyes are clear if my mind is of a slightly cynical bent having seen all this pseudoscience so many times in the past

 

[George]

Just a quick note, it's probably already been said but I've missed it. The WPI didn't "discover" this virus and they had HEAPS of help developing the assays to find it in blood samples. They started messing with it at Dr. Silverman's urging. They had Silverman's work with the virus and developing assays to to build on. Dr. Illa Sing also helped. Dr. Frank Russcetti who did major work with HIV and his wife Dr. Sara Russcetti who is still working on serology testing, also helped to design and refine the testing methods. It's not like the WPI just said 'oh, yeah we can find this, any body can. Just whip up the standard formula and voila'. They collaborated with some of the best minds on virology and cancer research. Dr. Mikovitz isn't a slouch when it comes to the science, though I think she may end up being a better spokesperson and promoter in the long run, And Dr. Lombardi has a decent vita himself. So a lot of really experienced people worked really hard to come up with the correct procedures and reagents and assays.


Even with all the work that came before it still took them from January of 09 to June of 09 just to figure it all out. Then from June of 09 to August of 09 to smooth out the papers edges. During that time the WPI collaborated with and talked to and dozens of people about techniques, ideals, and possibilities which turned out to greater good. And if it turns out that DR. SILVERMAN is right, because he's the one that was concerned about transmissiblity, then everyone will be thankful in the long run.

The First UK study was completed in under a month using standard methodology and considering Wessely involvement and all the press and hoopla might as well be tossed in the bin. The Groom et al study however, did try to find XMRV using a varity of methods. They just missed some really important aspects of the virus itself. It was done in a two month period and I get the impression that they didn't take the subtle points of the virus seriously. A virus that doesn't throw off quasi species is a really rare thing and I think that has a lot of people stumped.

I'm keeping my eye on the Dr. Kathryn Light study which looks promising and should be out any day and the Canadian Study.

Now that Koan has a new laptop I think we should make her pester Dr. Paul Jolicoeur over at The University of Montrea. (grins)

Just read Mithrial post, Duh on me! That was great Mith and way better worded!

 

[Advocate]

Hi George, Hi Gerwyn, Hi Mithriel, Hi ukxmrv, Hi Julius, Hi oerganix,

I just want you to know that your posts have given me a lift.

 

[usedtobeperkytina]

Mithrel, great post.

It is very clear. Some of the claims of what is supposed to be in publication is new to me and I don't know what the standard is, since I have no training or knowledge in such things.

Glad to have your input.

As has been stated, the UK studies, both of them, help us to know how not to find it. The problem was the Imp C study drew possibly erroneously conclusions.

This virus is different in so many ways.

And good point that Mikovitz didn't think to look into this virus. She was urged to study its possible relationship to CFS. We are talking so much about Mikovitz, but let's give Silverman an applause too.

But, Mikovitz, according to published reports, was able to see clearly from hearing Peterson talk about CFS and rates of cancer, that she came to him afterward and said, "That's a retrovirus."

She had tremendous experience with retroviruses and could see it fit the pattern.

All I can say is I am so glad we have something to debate. Just think of how much has changed since August of last year when all we did was complain and exchange the latest supplement that was supposed to help.

Tina

 

[jspotila]

Can anyone explain?

I was looking at one of the abstracts from last week's CROI meeting (see this thread for more from that meeting), and I have gotten myself confused. I've bolded the puzzler.

XMRV: Examination of Viral Kinetics, Tissue Tropism, and Serological Markers of Infection
X Qiu1, P Swanson1, K-C Luk1, J Das Gupta2, N Onlamoon3, R Silverman2, F Villinger3, S Devare1, G Schochetman1, and John Hackett, Jr*1
1Abbott Diagnostics, Abbott Park, IL, US; 2Cleveland Clin, OH, US; and 3Yerkes Natl Primate Res Ctr, Emory Univ, Atlanta, GA, US

Background: Xenotropic Murine Leukemia Virus-related Retrovirus (XMRV) is a human retrovirus recently discovered in familial prostate cancer tissue using DNA array based Virochip technology. Understanding viral replication kinetics, tissue tropism, and the host immune response is fundamental to establish the etiology of XMRV infection in human disease. Development of serologic assays to detect XMRV-specific antibodies would facilitate epidemiologic studies.

Methods: Five rhesus macaques were inoculated intravenously with XMRV. Blood was collected throughout the course of infection, and tissue from multiple organs was harvested at necropsy. Two macaques were necropsied at day 6 or 7 and one at day 144 post infection. The remaining 2 animals were re-inoculated with XMRV on day 158 and necropsied on day 291. XMRV-specific immunoreactivity was monitored by Western blot using viral lysate. Recombinant env gp70, p15E and gag p30 were utilized to develop serologic assays on the high-throughput automated ARCHITECT instrument system (Abbott Diagnostics).

Results: XMRV inoculation resulted in low transient plasma viremia, although proviral DNA persisted in circulating peripheral blood mononuclear cells for several weeks. Of interest, the earliest leukocyte targets were CD4+ T cells and NK cells followed by CD8+ enriched T and CD20+ enriched B cells (50% positive); CD14+ monocytes were negative. Animals sacrificed at the acute stage showed evidence of viral replication in spleen, lung, lymph nodes and liver. In contrast, sacrifice of 2 animals at 19 weeks post XMRV re-inoculation showed greater dissemination of XMRV DNA and RNA in various organs including the GI and urinary tract as well as in vaginal tissue of the one female. By Western blot analysis, all 3 chronically infected macaques developed antibody responses to env and gag proteins. The serologic assays demonstrated 100% sensitivity by detecting all Western blot positive serial bleeds from the XMRV-infected macaques. Preliminary results showed evidence of detectable reactivity to all 3 antigens in a low proportion (~0.1%) of US blood donors.

Conclusions: These data suggest that lymphocytes are a primary target for replication persistence (low grade replication) of XMRV in the absence of detectable plasma viremia. This study identified specific serological markers useful for detection of antibodies induced by XMRV infection. The prototype antibody assays will facilitate large-scale epidemiological studies.

So what does this mean? If they have specific serological markers, and those antibody assays can be used in high through-put technology, is the race over? I think the second UK study looked at antibodies but their assays were cross-reactive with non-XMRV antibodies, but that is not the case here? What has to happen for this study to be validated? And what does this mean in relation to WPI needing 4 blood draws to detect XMRV in many patients? Can one of the resident gurus unscramble this for me?

 

[Cort]

Dr. Mikovits has been answering email and she answered one of mine. We heard rumors that the CDC was up at the WPI- that was untrue but the WPI has shared it's reagants and antibodies, etc. with them. The CDC is apparently contacting other labs as well for their specimens and doing extensive testing.

Good News - Dr. Mikovits reported that the vaunted DHHS study is still,3 months later, in the 'design phase', which hardly suggests the government feels any urgency about it. Other agencies within the govt. are moving quickly. The CDC is acting on its own and Dr. Mikovits reported that she expects the NCI (National Cancer Institute) to PUBLISH SEVERAL PAPERS SOON both on XMRV in CFS and prostate cancer. Dr. Mikovits, of course, hails from the NCI.

The UK researchers didn't communicate with Dr. Mikovits or the WPI but its hard to imagine that she's not in touch with her former colleagues at the NCI researchers - given that her confidence is a good sign perhaps some positive papers are on the horizon. She also said that the "HIV docs get it 100%"

Bad News? The bad news is that I was contacted by a professional in the field who is very worried about XMRV right now and is not getting answers to his/her questions to the WPI. (Emails are not getting answered). This person outlined a scenario of contamination either at the Cleveland Clinic or the WPI. The contaminant is not an endogenous retrovirus but XMRV itself permeating all the samples.( I don't know how it doesn't get into the healthy controls). They were also worried about the efficacy of nested PCR - maybe someone can comment on that. They're also worried about the original cohort - specifically they're worried that the patients in it were selected on the basis of their positivity to another virus or for having T-cell rearrangements. Dr. Mikovits is not saying.

I asked Dr. Mikovits about the high rate of genetic homogenity in the early samples - something that suggests the samples came from one source - which could only be a contaminant.

She stated:

This was of course surprising to us given the prostate cancers were from Cleveland in different decades The contamination issue was settled in the original Science paper by the phylogenetic analysis and the immune response in the patients and the fact that XMRV was detected in samples sent to the NCI in Feb 2007 (before the WPI had a lab) ..these data satisfied the referees and were edited out of the final version of the manuscript as the editor as Science did not think it was important.

It seems to me that if the patients are having an immune response to the virus then that would take care of the idea that their blood samples had been contaminated with it????

I think we should be prepared for anything; great news or bad news. It is true that its nice to have something to get really fired up about - thats quite a difference.

 

[guest]

Thank you Cort, for posting this.

I have to throw in some more critics. In the last "This week in virology" podcast the virologists also talk about the second UK study and they point towards a possible contamination. I mean they are professors and really should know what they say. They were very interested in CFS and see it as a biological illness but now sceptisism takes over regarding the connection between CFS and XMRV.

You find it in the middle of the podcast.

http://www.virology.ws/2010/02/21/twiv-70-hacking-aphid-behavior/

 

[Hope123]

Cort, the part about T-cell rearrangements perhaps alludes to the number of lymphoma patients that Peterson talked about at CFSAC. My notes of course are jumbled with all the various talks recently but I recall there was either a WPI statement or Dr. M. saying that these were NOT part of the Science study although every one of the lymphoma patients were positive for XMRV. (Anyone else remember this?)

 

[garcia]

This person outlined a scenario of contamination either at the Cleveland Clinic or the WPI. The contaminant is not an endogenous retrovirus but XMRV itself permeating all the samples.

IMHO this isn't a realistic scenario. And surely it would have to be a contaminant at all 3 institutions at once (CC, WPI and NCI) since all the experiments were run independently at all 3 institutions?

( I don't know how it doesn't get into the healthy controls).

I think you've destroyed the argument with that one observation.

I asked Dr. Mikovits about the high rate of genetic homogenity in the early samples - something that suggests the samples came from one source - which could only be a contaminant.

I thought this issue has been dealt with. This is a very slow replicating virus. It is not that the WPI-isolated XMRV is homogeneous, its that all isolated XMRV is homogeneous (whether from prostate cancer, CFS etc.). If it was one contaminant you would expect the isolated viral sequences to be near identical, but there was enough difference in the genetic sequence to suggest we are not looking at a single contaminant, but a slow-replicating virus.

Also do we not think that someone like John Coffin, one of the greatest retrovirologists in the world hasn't thought of (and eliminated) this scenario?

I think we should be prepared for anything; great news or bad news.

A couple of weeks ago I might have agreed with you. But all the evidence seems to be pointing in the direction of XMRV being the cause of ME/CFS. The evidence also points to this being a difficult retrovirus to detect.

All this remains to be proven of course. But as was noted in the recent retroviorology conference, they still don't know how exactly HIV causes Aids, after all those billions of dollars, and decades of research.

 

[Gerwyn]

IMHO this isn't a realistic scenario. And surely it would have to be a contaminant at all 3 institutions at once (CC, WPI and NCI) since all the experiments were run independently at all 3 institutions?



I think you've destroyed the argument with that one observation.



I thought this issue has been dealt with. This is a very slow replicating virus. It is not that the WPI-isolated XMRV is homogeneous, its that all isolated XMRV is homogeneous (whether from prostate cancer, CFS etc.). If it was one contaminant you would expect the isolated viral sequences to be near identical, but there was enough difference in the genetic sequence to suggest we are not looking at a single contaminant, but a slow-replicating virus.

Also do we not think that someone like John Coffin, one of the greatest retrovirologists in the world hasn't thought of (and eliminated) this scenario?



A couple of weeks ago I might have agreed with you. But all the evidence seems to be pointing in the direction of XMRV being the cause of ME/CFS. The evidence also points to this being a difficult retrovirus to detect.

All this remains to be proven of course. But as I noted in the recent retroviorology conference, they still don't know how exactly HIV causes Aids, after all those billions of dollars, and decades of research.

I agree the contamination idea is highly unlikely when the simplest answer is that the tests in the English studies were not sensitive enough to detect it even if they had correct diagnosis which they did not.That final point is key they still dont know how the virus causes the condition.There are still virologists that use the term piggy back virus

 

[Esther12]

A couple of weeks ago I might have agreed with you. But all the evidence seems to be pointing in the direction of XMRV being the cause of ME/CFS. The evidence also points to this being a difficult retrovirus to detect.

All this remains to be proven of course. But as was noted in the recent retroviorology conference, they still don't know how exactly HIV causes Aids, after all those billions of dollars, and decades of research.

I don't know. The 97% figure from the WPI has always made me worry something odd is going on with their results, and the two failed replication attempts haven't boosted confidence.

I'd have always been amazed if a single retrovirus caused all canadian definition CFS cases - but thought XMRV was likely to play a part in a significant sub-section of cases, and hopefully encourage more serious research for the rest. I really don't know - it's so up in the air at the moment that my thoughts are probably more emotionally driven than evidence based. The WPI's overwhelming confidence slightly unsettles me too - hopefully they know about a lot of successful replication studies we're unaware of rather than just getting carried away with their own work.

 

[Gerwyn]

I don't know. The 97% figure from the WPI has always made me worry something odd is going on with their results, and the two failed replication attempts haven't boosted confidence.

I'd have always been amazed if a single retrovirus caused all canadian definition CFS cases - but thought XMRV was likely to play a part in a significant sub-section of cases, and hopefully encourage more serious research for the rest. I really don't know - it's so up in the air at the moment that my thoughts are probably more emotionally driven than evidence based. The WPI's overwhelming confidence slightly unsettles me too - hopefully they know about a lot of successful replication studies we're unaware of rather than just getting carried away with their own work.

The symptom pattern for aids can be very similar neuroendocrine autonomic immunological malaise fatigue etc that is all caused by one virus one way or another

 

[Esther12]

The symptom pattern for aids can be very similar neuroendocrine autonomic immunological malaise fatigue etc that is all caused by one virus one way or another

But the way CFS spreads seems pretty different.

 

[ukxmrv]

I'm just thinking out-loud here.

If the two UK studies show us "how not to do it", we are left to consider why no other UK establishment body (NHS, MRC or "Establishment" sources) have funded a study to show us "how to do it"?

Science doesn't operate in a vacuum.

It may be that a study to show us "how to do it" takes a lot longer to do and costs a great deal more money

It may be that these studies are not being funded in the UK. We do not know what is in the pipeline apart from the hints we find in the MEA web-pages etc. The Swedish study is not a replication.

It may be fair to argue that by ONLY funding "quick" research projects that are not attempts to replicate the WPI research that the "Establishment and quasi-government agencies" have either by a deliberate strategy, simple incompetence, a gamble or a calculated risk conspired to bury, discredit, or disprove the WPI results.

Are we the victims of scientific egos that wanted to prove that they could do "it", do "it" better than the WPI, funded by a range of sources who like the MRC not funded biomedical research before, or simple incompetence?

 

[Gerwyn]

But the way CFS spreads seems pretty different. I say this as someone whose partner also has CFS - so I'm certainly open to it being a retrovirus, but it does seem strange.

I agree .The mode of transmission and symptomology are of course different.there seems to be a pattern of cell cell transfer here which is another reason why it is so dashed difficult to detect.This mode of infective spread appears to be quite normal for a number of low titre viruses.There also seems to be a resevoir of viral particles in reproductive organs and fluids in primates and in semen in humans but I,m afraid thats all I know

 

[George]

Good Science Takes Time

(grins)

 

[oerganix]

Science does not include details of how the tests were done in original papers, these are available as supplementary information. My virologist friends feel that the details given in these by the WPI were very good and better than those seen in a lot of papers.

The reason for multiple samples is that XMRV does not replicate very often. It is only at those times the virus is replicating that it is free in the blood and able to be found by PCR. This means that only a sample taken at the right time will give a positive result unless you stimulate the cells in some way that encourages the virus to begin replication. Some patients needed blood taken at four separate times before the researchers caught the virus in the act of replicating.

When patients have been sending in blood to be tested XMRV is not being found in their blood unless it is actively replicating. If they culture the blood the virus gets coaxed into replicating so you get negative by PCR positive by culture.

Now they understand this they have stopped testing blood by PCR.

The WPI studies were done in more than one lab so they have already been replicated, that's why Mikovitz is confident that external replication will work if they follow the correct technique. This was a good, well done study, well planned and well executed.

The Kerr study was done quickly using things they already had to hand. They did not follow the WPI procedure because they did not want to do so. It wold have taken money and time to set up. There was no small deviation caused by a step left out of the Science paper, they made no attempt to follow the same procedure. This was not necessarily a bad thing, it might have worked but it was a decision they made, not something caused by sloppiness on the part of the WPI

They chose to use banked blood, probably also because of time and money constraints. Again it might have paid off but it didn't.

As for the Imp C study, school projects take longer to set up. This was rushed in the hope of putting a stop to research and the conclusions they reached were laughable if they were not so dangerous to us.

I stand by what I said. The Kerr study should have used a positive specimen of blood to test their procedure and only tested patients, banked blood or fresh, once they had perfected their technique. The fact that they didn't was a serious flaw.

I do not see why stating this means I am holding it up to a higher standard than the Mikovitz study. I am looking at both studies with a clear eye, I have a scientific background and virologists agree with me. It's time to let the WPI get on with their work for us.

Mithriel

Thank you Mithriel! I see your clear eye at work here and I really appreciate your insight and balance.

I also thank Gerwyn, who as usual, also has the 'clear eye' working very well.

Thanks to George for keeping us smiling, or laughing, and your great summaries.

Like Advocate, my spirits are raised by these posts and these posters. Whether XMRV is our boogyman or not, at last some progress is being made despite the denialists, the muddying of waters, the diversions via media copycat 'journalism', the incredibly cruel attempt to marginalize and minimalize us and our suffering.

I would also like to acknowledge the well-informed skeptics and even the cynics - you provide the resistance that brings out the best in the answers to your questioning.

It's a very hopeful time and I look forward to next month's revelations from Europe and the US.