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UPDATE: BLOOD WORKING GROUP

Megan

Senior Member
Messages
233
Location
Australia
... I hope they are doing a more broad sweep for (retro)viruses.

Couldn't agree more. One good thing about the retraction of the XMRV identification part of the paper, is it might allow more focus on other possibilities. And Lipkin's study sounds the right one to do that - I hope it still goes ahead. It has been evident for some time that the tests they used in the Science paper might have been picking up things other than XMRV.
 

eric_s

Senior Member
Messages
1,925
Location
Switzerland/Spain (Valencia)
But isn't the problem that whatever they have been picking up, now in the BWG they have found it more or less equally often in cases as in controls. And if they found a sample positive, when they got the same sample back with a different code they mostly could not correctly identify it as positive again. So as long as they can't prove they are able to tell cases from controls i don't see the possibility they are picking up something other than XMRV. Or at most something that's everywhere and sometimes it's there and sometimes not, but that doesn't make too much sense.

It will be interesting to hear from Dr. Hanson now. If she presents a blinded study that was positive i would like to hear what she has to say.
 

RustyJ

Contaminated Cell Line 'RustyJ'
Messages
1,200
Location
Mackay, Aust
But isn't the problem that whatever they have been picking up, now in the BWG they have found it more or less equally often in cases as in controls. And if they found a sample positive, when they got the same sample back with a different code they mostly could not correctly identify it as positive again. So as long as they can't prove they are able to tell cases from controls i don't see the possibility they are picking up something other than XMRV. Or at most something that's everywhere and sometimes it's there and sometimes not, but that doesn't make too much sense.

It will be interesting to hear from Dr. Hanson now. If she presents a blinded study that was positive i would like to hear what she has to say.

eric_s, Certainly we know that the other positives provided by Peterson would show up negative to the VP62 assay also, because they already had in the Levy/Peterson study.

All the BWG found is that patients provided by WPI and Peterson to the WPI did not have VP62. This is not the variant being picked up by WPI or Lo. The only person to pick up VP62 was Silverman.
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
eric_s, WPI did not use its assays, but was forced to use the BWG assay which supposedly could detect only VP62. Most probably most of the WPI patients submitted to the BWG did not have VP62 (if it actually is a HGRV). Certainly we know that the other positives provided by Peterson would show up negative to the VP62 assay also, because they already had in the Levy/Peterson study.

All the BWG found is that patients provided by WPI and Peterson to the WPI did not have VP62. This is not the variant being picked up by WPI or Lo. The only person to pick up VP62 was Silverman.

Why did the VP62 assay find it in positives and negatives. Who knows, perhaps it was contamination? But it is irrelevant.

Does anyone know where we can find information about the results and the methodology?

But isn't the problem that whatever they have been picking up, now in the BWG they have found it more or less equally often in cases as in controls. And if they found a sample positive, when they got the same sample back with a different code they mostly could not correctly identify it as positive again. So as long as they can't prove they are able to tell cases from controls i don't see the possibility they are picking up something other than XMRV. Or at most something that's everywhere and sometimes it's there and sometimes not, but that doesn't make too much sense.

It will be interesting to hear from Dr. Hanson now. If she presents a blinded study that was positive i would like to hear what she has to say.

Like Rusty says, I believe that the WPI were not allowed to use their own methodology in this study.
I don't know the details, but if that's the case then the results are quite meaningless.
 

Jemal

Senior Member
Messages
1,031
Does anyone know where we can find information about the results and the methodology?

I would say in the full Science article of the BWG study? I have not read it yet though, so I am not sure if it has these details.
 
Messages
13,774
Where are people getting this claim that the WPI weren't allowed to use their effective blood test, and could only use one which they did not think would work?

If true, this would be rather important. I don't see any reason to think that it is true though - it sounds unbelievable.
 

RustyJ

Contaminated Cell Line 'RustyJ'
Messages
1,200
Location
Mackay, Aust
Where are people getting this claim that the WPI weren't allowed to use their effective blood test, and could only use one which they did not think would work?

If true, this would be rather important. I don't see any reason to think that it is true though - it sounds unbelievable.

Some discussion about it prior to today, and certainly more detailed info today at mecfsforums - can't fault their analysis, even if it gets a bit rough over there.

I am sure Ecoclimber can provide access to paper.
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
I think it's helpful to remember that whatever happens to XMRV in relation to ME patients, XMRV is a very big story anyway.
It seems that XMRV is a man-made retrovirus, that contaminates labs all over the world, thus placing lab workers at risk of infection and vaccines at risk of contamination. This seems like a newsworthy story in itself. It's interesting that the newspapers aren't reporting this as a story in itself.


I've been doing some reading overnight, and things were not quite as I had thought there were.

Yes, there's been a partial retraction of the Science paper, and yes the BWG results were a failure.

But Judy's position has not changed massively.

I read the science article last night, and found that it is the best source of info so far.
It's a massive read (eight pages of A4), but the first three pages are very helpful.
I read it before I went to sleep, and I've totally forgotten all the details now, but Judy is still virus hunting and I think she believes that a very similar virus to XMRV is involved. I need to re-read it.

Here's some links that I've found for the science article.
http://www.mecfsforums.com/index.php?topic=9569.0

This link spits up the article, so you have to click on the subsequent links to read it all:
http://messages.finance.yahoo.com/S...m&bn=27865&tid=458900&mid=458900&tof=23&off=1

And this appears to be a PDF download:
http://www.google.co.uk/url?sa=t&so...XPzYgB&usg=AFQjCNHfoe0s4ppH8CnEnk5Z3Ivi-arg9Q




And here's an interesting post from villagelife on another thread:

http://phoenixrising.me/forums/showthread.php?13830-The-real-story-about-XMRV-coming-out-today

I saw this on facebook.....
(thanks to Lilly Meehan)

The big news that is lost in all the press about the BWG is that Silverman admitted that his lab had likely contaminated his part of the Lombardi study - which were the full sequences that he identified as XMRV and prompted the authors to use the term XMRV in the first place. Yet the rest of the Lombardi paper stands, as do findings by other labs of MLV-related viruses (MRVs) in CFS patients. The huge significance of this is that all this time researchers were looking for the wrong virus, when in reality patients from the Lombardi study had something similar but still distinct from XMRV. This helps explain both the negative studies and, likely, the BWG Phase III results. What we need the research community to do now is focus on sequencing isolates from patient blood all over again, to find out what virus or viruses many of us REALLY have.

So if this is correct, it seems that it's possible that the full sequences or isolate/s in the Science paper were due to contamination, but that the other findings in the Science paper were not due to contamination.

This could explain why there have been such disparate and mixed results all this time, with so many researchers unable to detect XMRV. Everyone was chasing the full isolates, which only exist as contamination.

So there is something there that Judy is detecting, but it might not be XMRV after all.

It's all really mind boggling. This could mean that Judy is not now chasing XMRV, but looking for similar viruses.

I'm not sure how convincing I find it all yet - it's too early to say.


From what I was led to believe earlier, there are still positive studies to be announced this weekend. But I'm not sure how significant these will be seen to be, now that there's been a partial retraction, and the BWG results were negative. And we are expecting Judy to present at this conference, so I expect that she will tell us what her position is and update us with her current research.
 

RustyJ

Contaminated Cell Line 'RustyJ'
Messages
1,200
Location
Mackay, Aust
I wonder how far back the knowledge that VP62 was a contaminant in the Silverman assay (could still be in some patients by the way) goes. For me there has been too much bashing with VP62 in the negative studies. WPI has tried to distance itself from VP62 for some time.

There has always been a bit of a problem for me with VP62 in prostate cancer. ME/CFS patients have neuro and immune irregularities, yet none of this showed up (or it wasn't discussed) in cases of prostate cancer and XMRV.

Ironically, one of the legitimate outcomes most likely from this is that any relationship between prostate cancer and XMRV should have been extinguished.
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
Where are people getting this claim that the WPI weren't allowed to use their effective blood test, and could only use one which they did not think would work?

If true, this would be rather important. I don't see any reason to think that it is true though - it sounds unbelievable.

I don't know any details about the assays that were used. I had assumed that the WPI could use their own.
But I seem to remember that the WPI could not culture their samples as they usually do, because the BWG needed to investigate reliable assays that did not need to use culturing (Because they can't culture millions of blood samples when working with the national blood bank.)
So I think that the WPI were not in full control of the methodology.
But it's all such vague memories for me now, that whatever I've said about it might not be accurate and should be checked elsewhere.
 

RustyJ

Contaminated Cell Line 'RustyJ'
Messages
1,200
Location
Mackay, Aust
It's all really mind boggling. This could mean that Judy is not now chasing XMRV, but similar viruses. I can't be sure about this though, as I've only been picking up bits and pieces.

You are almost correct Bob. It is the same virus, just variations that cannot be picked up by the VP62 assay. VP62 assay has three big fails:
  • It can only detect narrow selection of XMRV
  • Usual detection problems of finding XMRV in blood, when everyone knows, even the macaques, that the virus retreats to tissue reservoirs
  • And of course it really only picks up VP62, which probably doesn't exist except as a lab artifact


In fact it was Silverman who found XMRV and named it in prostate cancer. WPI found variations of the same virus, and in the absence of full sequences had to name it XMRV. At the time it was not recognized as a variation to the Silverman XMRV. But since the original Science paper, WPI have steadfastly stated that they are picking up all sorts of poly MLVs etc (just not VP62), just as Lo did. So the Lo study really was a replication of WPI.

I really hope the VP62 thing is understood by everyone reading these posts. I don't know how to explain it better. It is important not to lose hope in this. XMRV, or least variations of it, is still alive.

Thanks for the references Bob. I was too tired to pull it together.
 
Messages
13,774
I don't know any details about the assays that were used. I had assumed that the WPI could use their own.
But I seem to remember that the WPI could not culture their samples as they usually do, because the BWG needed to investigate reliable assays that did not need to use culturing (Because they can't culture millions of blood samples when working with the national blood bank.)
So I think that the WPI were not in full control of the methodology.
But it's all such vague memories for me now, that whatever I've said about it might not be accurate and should be checked elsewhere.

But if the WPI thought that they needed to culture their samples for accurate detection, why would they ever agree to a study where they thought they'd fail? They could have done one set of testing without culture, and one with.

I've not seen any official report about the WPI not being able to use their effective test either, so the whole thing could just be a rumour.
 
Messages
13,774
From the Science editorial:

In late August, the Blood Working Group completed its roughly $500,000 study, which conclusively determined that no one need worry about XMRV or MLVs in the blood supply. The nine labswhich included WPI, Ruscetti, and Lo at FDAeach had received blinded samples from 15 negative controls and 15 others who had tested positive for a gammaretrovirus in Lombardi et al. or Lo-Alter. Different teams cultured the virus, looked for antibodies to it, and used PCR to fish for DNA. All labs could use whatever assays they chose.



Only WPI and Ruscetti found intermittent evidence of viruses or antibodies in patients, but they also reported similar numbers of positive responses in the negative controls. What's more, there was no agreement between the two labs on which patient samples tested positive. What this says, at the very minimum, is that we can't find it reliably in the blood of patients we found it in before, Ruscetti says.
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)


You are almost correct Bob. It is the same virus, just variations that cannot be picked up by the VP62 assay. VP62 assay has three big fails:
  • It can only detect narrow selection of XMRV
  • Usual detection problems of finding XMRV in blood, when everyone knows, even the macaques, that the virus retreats to tissue reservoirs
  • And of course it really only picks up VP62, which probably doesn't exist except as a lab artifact


In fact it was Silverman who found XMRV and named it in prostate cancer. WPI found variations of the same virus, and in the absence of full sequences had to name it XMRV. At the time it was not recognized as a variation to the Silverman XMRV. But since the original Science paper, WPI have steadfastly stated that they are picking up all sorts of poly MLVs etc (just not VP62), just as Lo did. So the Lo study really was a replication of WPI.

I really hope the VP62 thing is understood by everyone reading these posts. I don't know how to explain it better. It is important not to lose hope in this. XMRV, or least variations of it, is still alive.

Thanks Rusty, that's very helpful.

Your VP62 explanation makes perfect sense to me.

The WPI have indeed been claiming for ages that they've been finding various HGRV's and different variations of XMRVs.
 

RustyJ

Contaminated Cell Line 'RustyJ'
Messages
1,200
Location
Mackay, Aust
But if the WPI thought that they needed to culture their samples for accurate detection, why would they ever agree to a study where they thought they'd fail? They could have done one set of testing without culture, and one with.

I've not seen any official report about the WPI not being able to use their effective test either, so the whole thing could just be a rumour.

WPI could not politically afford to walk away - they would have been ridiculed and sidelined from the scientific debate. It seems to have been a calculated gamble. Wear the Silverman retraction, ride out the negative BWG findings, but run with the core point: Not one study has faulted WPI's or Mikovits' work. That is something to build on.

Personally, I don't think they anticipated the Peterson/Levy thing which has basically sent everything off the rails. And of course Peterson/Levy was a big spanner in the works for the outcome of BWG.
 
Messages
13,774
WPI could not politically afford to walk away - they would have been ridiculed and sidelined from the scientific debate. It seems to have been a calculated gamble. Wear the Silverman retraction, ride out the negative BWG findings, but run with the core point: Not one study has faulted WPI's or Mikovits' work. That is something to build on.

Personally, I don't think they anticipated the Peterson/Levy thing which has basically sent everything off the rails. And of course Peterson/Levy was a big spanner in the works for the outcome of BWG.

Surely they could have said: We can only participate if we are able to use the testing methods we believe are most effective. I don't think that would have excluded them from scientific debate.

I don't know if it's accurate, but the quote I posted from the Science editorial above says that culturing was used, and that: "All labs could use whatever assays they chose."

This could be wrong (there have been plenty of misleading claims made about CFS previously), but we need some good reason to think that it's wrong before just making that assumption.
 

RustyJ

Contaminated Cell Line 'RustyJ'
Messages
1,200
Location
Mackay, Aust
From the Science editorial:

Esther, on going back over the posts at mecfsforums, I still can't confirm this. However it does appear that everyone except WPI used VP62 primers. So I should modify some of my posts.

WPi may have used their own primers which is why they picked up some positives. They also picked up negative controls as positive (probably because they were positive) having been declared negative by the inadequate VP62 primers.

So if WPI used their assays why didn't they pick up more positives? Not sure.


Here is a post from ISO on mecfsforums explaining the anomaly.
Little run down for you all.

In this study the WPI found 2 plasma gag samples positive. Both were from the negative controls.

These 2 were sequenced and are 1 to 3 nucleotides different to VP62, which makes them the same type Lo et al found.

"However, two plasma clinical aliquots were reported as positive in the WPI nested RT-PCR gag assay. These samples were from two different negative controls, and only one out of the three replicates was positive in each case. Sequencing of the excised bands revealed 1-3 base changes compared to XMRV derived from 22Rv1 (supporting online text)."


Ruscetti didn't take part in this section of the study and Lo change assay, to one using VP62 primers.

If others used VP62 to validate plasmas as negative, they would not have picked up anything because plasma RNA has sequence variability. Which explains why Lo cannot now detect the type of virus found in Lo et al. So the VP62 assays could not find integrated MLVs. (not the mouse sort) What they can find is free floating virions in plasma.

As the WPI use lower annealing temperatures their primers would pick up both.

So I would ask, were the plasmas validated negative using VP62 as positive controls to optimise the PCR and did those assays use high annealing temperatures? because using a free floating clone, which does not exist in nature, will never give a PCR the clinical sensitivity to pick up a methylated provirus.

High stringency primers will also not detect virion RNA in plasma because, as shown by the macaque studies, the RNA has sequence variability.
 

Jemal

Senior Member
Messages
1,031
I think Bob's analysis is correct, I had the same thoughts yesterday. That's why I said on this thread the WPI will be able to reinvent XMRV. Others might as well...
The WPI still found something in the Science study and that's why it hasn't been fully retracted yet and it could also explain a lot of the confusion. Science might still fully retract the WPI paper as well, by the way, they seem to be looking into it.

No official WPI statement so far I think, besides a few snippets in several articles. I am very interested in what they have to say. I have the feeling they will not back down from this avenue of research.
 
Messages
13,774
@ RustyJ: But the Science editorial said that culturing was used, and that labs could use whatever assay they wanted. I've not yet seen any reason to think this is wrong. If the WPI weren't able to use their effective testing, then that would be a really important fact, but so far it just seems to be an internet rumour. Does anyone have any official source claiming that labs were prevented from using their most effective testing techniques?

@ Jemal: I don't think that we can still say that the Science article did find anything. If their testing does not hold up under blinded conditions, then that means that there probably was some error.

To me, it looks like we're back to square one. Maybe some murine related retro-virsues are related to CFS, but I don't think that we have any more reason for thinking so now than we did in 2008 - probably less.
 

RustyJ

Contaminated Cell Line 'RustyJ'
Messages
1,200
Location
Mackay, Aust
@ RustyJ: But the Science editorial said that culturing was used, and that labs could use whatever assay they wanted. I've not yet seen any reason to think this is wrong. If the WPI weren't able to use their effective testing, then that would be a really important fact, but so far it just seems to be an internet rumour. Does anyone have any official source claiming that labs were prevented from using their most effective testing techniques?

@ Jemal: I don't think that we can still say that the Science article did find anything. If their testing does not hold up under blinded conditions, then that means that there probably was some error.

To me, it looks like we're back to square one. Maybe some murine related retro-virsues are related to CFS, but I don't think that we have any more reason for thinking so now than we did in 2008 - probably less.

Where I got mixed up was WPI did not validate the negative controls (whereas I stated WPI essentially did not use their primers for the positives and negatives.) The negative controls could have been positive (it appears they were only tested by PCR) and even the WPI PCR only picks up 30%. This is why WPi picked up positives in the negative controls when it came to testing them in the blinded tests.

So the negative controls definitely were not cultured for validation. Still pouring over the data to find out why they didn't pick up more positives and if they cultured.