XMRV CFS UK study #II

[CBS]

Nagging thoughts

It's been a few days and I'm having nagging thoughts regarding the new Groom XMRV study:

1. The latest Groom et al. study found a background level of XMRV similar to that of several US studies (4%) in the blood of their 'healthy controls.'
2. Groom et al. did not find that same background level of XMRV in their CFS patients. Why not?
3. I do not see it stated explicitly in the Groom paper but it appears that the CFS blood samples were stored for 1.5-9.0 years where as the 'healthy control' blood was fresh (can anyone verify or dispute this?). The WPI CFS samples were even older in some cases.
4. Groom et al. appears to have left out a step that the WPI deems necessary:
   

   "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."

5. If XMRV, when present, is there in such low numbers and improper storage degrades the ability to detect XMRV, would this account for Groom et al. being able to find a background rate in healthy controls similar to the other US studies while finding nearly nothing in the CFS patient's stored blood (in both groups without the advantage of growing the white blood cells)?

Could the issue here be - that low copy numbers and potential degradation in the storage process makes the need to grow the white blood cells before looking for XMRV in stored samples more critical than when looking for XMRV in 'fresh' blood?

 

[Gerwyn]

Leaving aside the motivations of a research team, it seems irresponsible to not FIRST try and replicate the findings. There is plenty of time for validation studies, but the first thing that should have happened is somone trying to replicate the study to see if they got similar results.

One of the first things you learn is science class as a kid about scientific experiments is to control as much as possible. It's a no brainer that the very first thing that should have been done is to see if someone else can get similar results using the same protocols.

I can't believe we are still waiting for someone to make this simple step forward in this research.

We can debate all day which protocols are better and the value of validation studies and the significance of other testing methods not detecting XMRV in PWCs, but my gosh, can someone please do the only logical next step in this research and try and replicate the WPIs finding using THEIR protocols and some of their samples?

It seems that following the simple scientific method is beyond some peoplel .It genuinely baffles me because as you say it is one of the first things you are taught as a science student.The first step is to replicate using the same protocol then see if your protocol gets the same results in the same conditions then try your protocol in different conditions -It as basic science and an abs absolute no brainer----These professional scientists must know this so why are they not doing it

 

[Gerwyn]

It's been a few days and I'm having nagging thoughts regarding the new Groom XMRV study:

1. The latest Groom et al. study found a background level of XMRV similar to that of several US studies (4%) in the blood of their 'healthy controls.'
2. Groom et al. did not find that same background level of XMRV in their CFS patients. Why not?
3. I do not see it stated explicitly in the Groom paper but it appears that the CFS blood samples were stored for 1.5-9.0 years where as the 'healthy control' blood was fresh (can anyone verify or dispute this?). The WPI CFS samples were even older in some cases.
4. Groom et al. appears to have left out a step that the WPI deems necessary:
   
5. If XMRV, when present, is there in such low numbers and improper storage degrades the ability to detect XMRV, would this account for Groom et al. being able to find a background rate in healthy controls similar to the other US studies while finding nearly nothing in the CFS patient's stored blood (in both groups without the advantage of growing the white blood cells)?

Could the issue here be - that low copy numbers and potential degradation in the storage process makes the need to grow the white blood cells before looking for XMRV in stored samples more critical than when looking for XMRV in 'fresh' blood?

yes absolutely

 

[George]

Hey Shane

Couple of points, yep the second study did use older blood. Buttttttt. . . The WPI didn't, sort of. (grins)

Turns out that the blood used for the WPI study was fresh, and from 4 separate draws. Now this is not to be confused with patient 1125 who was from a stored sample that was taken out and "cultured" to raise the level of copies of XMRV per uL and where included in the Science paper.

Dr. Mikovitz makes a statement (see Advocates Repost of LadybugMandys post, it's number #390) that they should not have included unpublished data in the Media.

So you pretty much summed up the problem right there. Old blood from CFS patients that was cultured for only 18 hours rather than, I think it's 10 days, (that could be wrong) and then fresh blood from the healthy controls.

It's still a well designed study they just didn't find any XMRV. Although they almost stumbled upon it by accident. (grins) But it was done way back in November so it could well be that when the study was designed they didn't understand the nature of our particular and somewhat peculiar penguin.

 

[flex]

That could be a really important statement!

George

this is the closest I can find so far.

"Chronic fatigue syndrome affects a large number of people and our findings are likely to be very disappointing to these patients, their families and their friends. It is important that we keep an open mind about new scientific discoveries which point to possible causes of this often very serious condition. Replication is an important part of the scientific method and, as the initial findings have not yet been replicated, I think it will be important to develop standardised samples and assays for XMRV that can be rapidly tested by different laboratories around the world."

Kate Bishop


No mention, of neurological disease but obviously, Kerr has made his understanding of ME clear in his previous writings. Would it make sense for someone to email Kerr and pose all these questions we are discussing here.



Retrovirology 2010, 7:10 doi:10.1186/1742-4690-7-10
Harriet C T Groom (hgroom@nimr.mrc.ac.uk)
Virginie C Boucherit (vbouche@nimr.mrc.ac.uk)
Kerry Makinson (k.makinson@sgul.ac.uk)
Edward Randal (erandal@sgul.ac.uk)
Sarah Baptista (m0705047@sgul.ac.uk)
Suzanne Hagan (Suzanne.Hagan@gcal.ac.uk)
John W Gow (John.Gow@gcal.ac.uk)
Frank M Mattes (frank.mattes@bartsandthelondon.nhs.uk)
Judith Breuer (j.breuer@ucl.ac.uk)
Jonathan R Kerr (jkerr@sgul.ac.uk)
Jonathan P Stoye (jstoye@nimr.mrc.ac.uk)
Kate N Bishop (kbishop@nimr.mrc.ac.uk)
ISSN 1742-4690
Article type Research
Submission date 11 January 2010
Acceptance date 15 February 2010
Publication date 15 February 2010
Article URL

 

[anne]

Wait a second.

How did they find XMRV in the controls? Their method or the WPIs?
Does this mean they actually CAN find XMRV?

So, if the CFS blood had been fresh, we might have had a useful study--that is, if they could find XMRV, they would either find it at the same rate as the healthy controls, or something significantly higher?

And--okay, I'm no scientist. But if you find a virus in your healthy controls and not in your test sample, don't you then step back and say to yourself, "Self? That's weird. What's different about these samples?"

I mean, no one is arguing that CFS suppresses XMRV (though maybe that's a side effect of the abnormal illness beliefs. You are so damned healthy you can't even get a retrovirus.) So there's got to be something else. And if that something else is in your collection process, don't you correct it? Isn't the failure to do so a little...careless?

 

[flex]

And--okay, I'm no scientist. But if you find a virus in your healthy controls and not in your test sample, don't you then step back and say to yourself, "Self? That's weird. What's different about these samples?"

I mean, no one is arguing that CFS suppresses XMRV (though maybe that's a side effect of the abnormal illness beliefs. You are so damned healthy you can't even get a retrovirus.) So there's got to be something else.

This is the funniest comment I've read here in a long time.

 

[George]

Wait a second.

How did they find XMRV in the controls? Their method or the WPIs?
Does this mean they actually CAN find XMRV?

So, if the CFS blood had been fresh, we might have had a useful study--that is, if they could find XMRV, they would either find it at the same rate as the healthy controls, or something significantly higher?

And--okay, I'm no scientist. But if you find a virus in your healthy controls and not in your test sample, don't you then step back and say to yourself, "Self? That's weird. What's different about these samples?"

I mean, no one is arguing that CFS suppresses XMRV (though maybe that's a side effect of the abnormal illness beliefs. You are so damned healthy you can't even get a retrovirus.) So there's got to be something else. And if that something else is in your collection process, don't you correct it? Isn't the failure to do so a little...careless?

Now that I'm through laughing my poor tail off. That was a picture perfect statement anne. Thank you.

To answer your question, they made up an antibody test that tested for things that would inhibit XMRV it was kinda strange test cause it would also inhibit MLV and Friend MV and another one that I forget. And what do you know 25 of the healthy controls popped up positive and one (old blooded) CFS person. but then they messed around with that and decide that only 4 of the 26 (which would have been 4% by the way, go figure!!!) really really, might have actually, maybe had XMRV.

 

[George]

Flex
Thanks so much for digging that up.

 

[Gerwyn]

Flex
Thanks so much for digging that up.

Lets transfect these cells with what we assume will be an infectious virus for 18 hours when all the published work says that it will take 14 days for these cells to produce new virus and then test to see if we have the virus no how strange!!

 

[Cort]

My understanding is that freezing does break up viruses but that enough usually remains for them to be found afterwards. Perhaps there's simply too little of the virus present for enough of it to survive freezing.

 

[cfs since 1998]

My understanding is that freezing does break up viruses but that enough usually remains for them to be found afterwards. Perhaps there's simply too little of the virus present for enough of it to survive freezing.

Didn't DeFreitas insist that her virus couldn't survive freezing, but the labs trying to replicate her work used frozen samples anyway?

 

[Cort]

Tom Folks at the CDC said he eventually tried everything; frozen and fresh - and still couldn't find it.

I just checked the Science paper - the WPI used frozen samples for all their work.

 

[kurt]

Couple of points, yep the second study did use older blood. Buttttttt. . . The WPI didn't, sort of. (grins)

Turns out that the blood used for the WPI study was fresh, and from 4 separate draws. Now this is not to be confused with patient 1125 who was from a stored sample that was taken out and "cultured" to raise the level of copies of XMRV per uL and where included in the Science paper.

Dr. Mikovitz makes a statement (see Advocates Repost of LadybugMandys post, it's number #390) that they should not have included unpublished data in the Media.

So you pretty much summed up the problem right there. Old blood from CFS patients that was cultured for only 18 hours rather than, I think it's 10 days, (that could be wrong) and then fresh blood from the healthy controls.

It's still a well designed study they just didn't find any XMRV. Although they almost stumbled upon it by accident. (grins) But it was done way back in November so it could well be that when the study was designed they didn't understand the nature of our particular and somewhat peculiar penguin.

That 10+ days was for the culture study only, to produce proteins, and not for the original PCR. At least not that WPI has told us...

Also, if WPI had to take up to four blood samples to find XMRV in their CFS group, did they also look that hard for XMRV in the controls? Did they also take four blood samples from each control? If not, that raises questions and Mikovitz should explain what really happened in the study in much more detail. IF this is really a private study and they will only give details to their direct collaborators, and not the public scientific community, they should say so.

I think it is high time for more details from WPI, maybe another publication, they have had enough time, what are they studying now?

Regarding fresh vs frozen, if scientists can extract DNA from up to 500 million year-old fossils (yes, that was not a typo), and WPI was able to extract from frozen CFS sample, this is really a non-issue. The larger problem I see is the problem with WPI revealing important details piece-meal, only as others are having problems.

Raul J. Cano and Monica K. Borucki have discovered and have actually revived (brought back to life!) over 1,000 types of bacteria and other microorganisms. Some of the life-forms date as far back as 135 million years which was the time of the dinosaurs. - http://www.mhrc.net/ancientDNA.htm (another example given was bacteria revived from salt, dated 250-500 million years old)

 

[Cort]

Also, if WPI had to take up to four blood samples to find XMRV in their CFS group, did they also look that hard for XMRV in the controls? Did they also take four blood samples from each control?

Things are getting a little messy here. I quickly looked at the Science paper and they didn't appear to say that they did such extensive testing - up to four stabs plus from up to four different time points (plus the need to culture!). I would imagine that that's quite unusual in a viral study and honestly, if that's what it took and they didn't state that - then they're changing the rules in the middle of the game - and costing researchers a ton of money by waiting so long to tell everyone. If the virus is THAT hard to find (culturing plus multiple time points and multiple stabs at it) then they also shot themselves in the foot by not stating so. (Unless that info is actually in the Science paper and I couldn't find it).

My guess is that the virus more readily showed up in that first probably quite sick group and that its been much harder to find in more typical CFS patients and they're learning that and we're learning that as we go along.

 

[Gerwyn]

That 10+ days was for the culture study only, to produce proteins, and not for the original PCR. At least not that WPI has told us...

Also, if WPI had to take up to four blood samples to find XMRV in their CFS group, did they also look that hard for XMRV in the controls? Did they also take four blood samples from each control? If not, that raises questions and Mikovitz should explain what really happened in the study in much more detail. IF this is really a private study and they will only give details to their direct collaborators, and not the public scientific community, they should say so.

I think it is high time for more details from WPI, maybe another publication, they have had enough time, what are they studying now?

Regarding fresh vs frozen, if scientists can extract DNA from up to 500 million year-old fossils (yes, that was not a typo), and WPI was able to extract from frozen CFS sample, this is really a non-issue. The larger problem I see is the problem with WPI revealing important details piece-meal, only as others are having problems.

The WPI methodology is not perfect but when looking at virus recovery from whole blood you must consider infectivity half life and the effect of rethawing on an already miniscule viral load sorry age is an issue do you know how many pCR amplification cycles it takes to detect latent epstein Barr35 yet the british study tried one replication cycle The contol group had massively elevated endogenousretrovirus expression the cells used for transfection need 14 days to produce virus yet they harvested the"virus" after 18hours and used non specific neutralisation.

The link you posted was to a CREATIONIST organisation the DNA recovered was fragmentory the back to life bit was creationist spin

 

[julius]

I was just reading your blog, Cort, and it brought up a question. I'm not sure if it has been asked before, but there hasn't been any great discussion on it.

What technique did the Japanese use? They looked at blood, and they did it the summer before the WPI study was published.

Has anyone else tried using their technique?

Does anybody even know what the technique was?

 

[kurt]

The WPI methodology is not perfect but when looking at virus recovery from whole blood you must consider infectivity half life and the effect of rethawing on an already miniscule viral load sorry age is an issue do you know how many pCR amplification cycles it takes to detect latent epstein Barr35 yet the british study tried one replication cycle The contol group had massively elevated endogenousretrovirus expression the cells used for transfection need 14 days to produce virus yet they harvested the"virus" after 18hours and used non specific neutralisation.

How can age be an issue for replication/validation studies only and not for WPI? I just don't buy that. Also, you seem to have more background in PCR testing, isn't it true that the labs first must do the extraction of the DNA, before running the PCR, and in that process will learn whether or not they have a viable DNA sample? So I assume that any competent PCR lab could quickly tell if they had testable DNA or not. Don't they have a way to control for viability of the DNA?

The link you posted was to a CREATIONIST organisation the DNA recovered was fragmentory the back to life bit was creationist spin

I did not notice the source or that those claims were spin-doctored, thanks for pointing that out, but bringing an old bug back to life was not the primary point. So spin or not, the fact is that very old blood can still produce testable DNA.

 

[Bob]

Professor Malcolm Hooper's opinion of the UK research

We didn't get explicit permission to quote Prof Malcolm Hooper, so I won't quote him directly,
but you can now read his quote on an MEActionUK webpage, here:
http://www.meactionuk.org.uk/xmrv-research-and-the-ramsay-research-fund.htm

(search for the word 'skulduggery', and you'll find his quote)

 

[Gerwyn]

How can age be an issue for replication/validation studies only and not for WPI? I just don't buy that. Also, you seem to have more background in PCR testing, isn't it true that the labs first must do the extraction of the DNA, before running the PCR, and in that process will learn whether or not they have a viable DNA sample? So I assume that any competent PCR lab could quickly tell if they had testable DNA or not. Don't they have a way to control for viability of the DNA?



I did not notice the source or that those claims were spin-doctored, thanks for pointing that out, but bringing an old bug back to life was not the primary point. So spin or not, the fact is that very old blood can still produce testable DNA.

testable is not viable and here you are looking for a very small specific sequence the wpi did not use whole blood they concentrated their protien pre assay from specific cells and further amplified the virus load with culture plus the british blood was taken by phlebotomists with no idea how much care you need to take if you are trying to isolate virus from blood they cant have known they were looking for anything in particular they were ordinary blood samples for routine tests in the uk lysis is a huge problem
the british studies were looking for virus in normal titres