Article: From the PCR Side: the Cooperative Diagnostics XMRV Interview with Dr. Brent Satterfield

Article: From the PCR Side: the Cooperative Diagnostics XMRV Interview with Dr. Brent

You can view the page at http://forums.aboutmecfs.org/content.php?376-From-the-PCR-Side-the-Cooperative-Diagnostics-XMRV-CFS-ME-CFS-Interview-with-Dr.-Brent-Satterfield

 

Posted by Cort:

For instance if you take samples of HIV from just two different people you've find scads of variability. In fact, if you take a sample of HIV from the same person at different points in time you'll lots of variability.

However if you take XMRV from several people even if they live far apart from each other - which probably means the virus had to go through other people to wind up in both - the virus is virtually the same.

The following quote from this article in 'Nature' makes the point that there is little genetic variability of the other human retrovirus HTLV. Just because something is a retrovirus it doesn't necessarily mean it should behave like HIV. HTLV is obviously very different to HIV in a number of ways, lack of genetic variability being one of them.

http://www.nature.com/onc/journal/v24/n39/full/1208968a.html

Despite the frequent error rate of retroviral replication and high levels of provirus in infected lymphocytes, HTLV-I has relatively low intra- and inter-individual sequence variability (Gessain et al., 1992). This apparent paradox has been postulated to be due to the clonal expansion of HTLV-I-infected lymphocytes (Wattel et al., 1995). After a brief period of reverse transcriptase-mediated replication soon after initial infection, multiplication of provirus occurs mainly via clonal expansion of the infected lymphocyte rather than production of new virions.

 

I asked Satterfield about this on the phone. My recollection is that he does believe that it is picking up evidence of infection. He does not believe that is picking up evidence of a different MLV infection but that it's probably picking another virus. It is a very interesting finding.

Again there is the question, though, why other groups which are also looking for antibodies against MLV's have not found them yet in CFS patients.

Cort, thanks for your response. But I still don't understand why Satterfield would rule out other MLV's - is it because of the other negative studies? Regardless of what one thinks of the 22Rv1 contamination theory, this still leaves Lo and Alter's findings untouched, Hanson claims to have found similar MLV's and it is also clear that the WPI's tests would have picked these up too? Both WPI and Alter's group have run the mtDNA test to exclude contamination and Alter went out of his way at the Dec BPAC meeting to make sure the transript recorded that they had alo run the IAP test on their samples too.

Also re the antibody tests, I thought Rachel Bagni at the NCI was getting positive antibody responses for the Lo and Alter samples. Again this would seem supportive of an MLV finding.

 

Cort said

The Lombardi paper stated the strains they found were essentially the same as VP62 and, I believe VP34 - which later research showed were essentially the same as 22RVI.

As I have already pointed out on reply 24 above, Lombardi found VP62, VP35 and VP42, and since those papers were published Mikovits has stated that she's revisited the samples and found greater variability.


Kurt

Gerwyn's comment as quoted also contains a serious factual error, they did not base their test on 62ul of plasma. Did you even read the article at the start of this thread? Here is a quote from the article that answers most of the points being made about the sample volume.

Kurt, we've dealt with this before. Here are details of PCR perameters from Satterfield's paper, table two:

http://forums.aboutmecfs.org/showth...-study-is-this-new-looks-to-be-from-CDC/page4

 

Is it just me or does this whole article and the resulting responses by Cort & Kurt sound more like a Public Relations exercise than anything else? It reminds me of the CAA and their attempts to silence their critics.

I ask the question again to Kurt Rowley:
Will Cooperative Diagnostics be refunding the money they took from patients for a pin-prick test which could not possibly work and which never found a single positive? (one that even Dr Coffin derided at the CFSAC meeting in 2009)

 

Posted by Cort:


The following quote from this article in 'Nature' makes the point that there is little genetic variability of the other human retrovirus HTLV. Just because something is a retrovirus it doesn't necessarily mean it should behave like HIV. HTLV is obviously very different to HIV in a number of ways, lack of genetic variability being one of them.

http://www.nature.com/onc/journal/v24/n39/full/1208968a.html

Despite the frequent error rate of retroviral replication and high levels of provirus in infected lymphocytes, HTLV-I has relatively low intra- and inter-individual sequence variability (Gessain et al., 1992). This apparent paradox has been postulated to be due to the clonal expansion of HTLV-I-infected lymphocytes (Wattel et al., 1995). After a brief period of reverse transcriptase-mediated replication soon after initial infection, multiplication of provirus occurs mainly via clonal expansion of the infected lymphocyte rather than production of new virions.

That's very interesting....Clonal expansion - apparently. instead of replicating, the virus is causing the lymphocytes to split and that's how it survives and spreads through the body. Fascinating!

It sounds like we can say that we just don't know what the low genetic variability means. All we can say right now is that the genetic clustering of the XMRV strains around the 22RV1 strain is of concern. Some researchers did note that it's possible that XMRV has replicated and therefore changed more in the lab than it has in the body - which is a fascinating idea. Maybe XMRV is almost completely shot down in the body...it can get in there initially and infect cells - but the immune system pretty much hammers it - so there is little activity other than clonal expansion and few genetic changes.

What I think we really need is for the WPI to release or publish their variability findings. If they're having trouble publishing they could concievably just release them on their website or on the IACFS/ME website.

On the other hand, since they and others are able to grow XMRV in cells in the lab I would think somebody should be seeing this clonal expansion? Ie they should be able to put XMRV in a culture of white blood cells and then, after a time, count the white blood cells and see if the number has gone up? (or has not gone down as expected) thereby indicated XMRV is prompting the WBC's to replicate. I'm sure its not as easy as that but its an idea.

 

Cort, thanks for your response. But I still don't understand why Satterfield would rule out other MLV's - is it because of the other negative studies? Regardless of what one thinks of the 22Rv1 contamination theory, this still leaves Lo and Alter's findings untouched, Hanson claims to have found similar MLV's and it is also clear that the WPI's tests would have picked these up too? Both WPI and Alter's group have run the mtDNA test to exclude contamination and Alter went out of his way at the Dec BPAC meeting to make sure the transript recorded that they had alo run the IAP test on their samples too.

Also re the antibody tests, I thought Rachel Bagni at the NCI was getting positive antibody responses for the Lo and Alter samples. Again this would seem supportive of an MLV finding.

My take is that Satterfield is not confident about the MLV findings - he believes they look too much like mouse endogenous retroviruses and not enough like XMRV. Lo/Alter appeared to me at least to have done eveything they could to rule out contamination but the sequences are so much like endogenous retroviruses that I believe that what Satterfield wants is that they grow the viruses (proving they are from viruses) or show DNA integration into human DNA for more researchers will come around. From talking with Dusty Miller he believes the same.

Their findings are, on the other hand, untouched by the 22RVI controversy - which is what is hammering XMRV right now. The 22RV1 cell line does not produce MLV's like that - it produces XMRV.

Yes, the news Dr. Mikovits reported from Rachel Bagni was very hopeful. Hopefully it will either get into a conference proceeding or better yet, a study. Validated antibody findings is another way to turn this thing around. The WPI could use her antibody assay with the BWG (I thought it was from XMRV) showing they can find XMRV in their patients - and we'd be off to the races again.

 

Is it just me or does this whole article and the resulting responses by Cort & Kurt sound more like a Public Relations exercise than anything else? It reminds me of the CAA and their attempts to silence their critics.

I ask the question again to Kurt Rowley:
Will Cooperative Diagnostics be refunding the money they took from patients for a pin-prick test which could not possibly work and which never found a single positive? (one that even Dr Coffin derided at the CFSAC meeting in 2009)

It may sound that way to you Garcia but I assure you that for me - its just trying to understand the science. It just happens not be mostly supporting XMRV at this point IMO. That could change tomorrow.

As I said earlier - I believe Cooperative Diagnostics would be obligated to do that if their later test results indicated that their earlier test results were wrong. I believe VIP Dx to their credit went back and redid earlier negative test results using their new protocols but since CD's later test didn't suggest the earlier test was inaccurate there was no reason to do that.

Of course they wouldn't know that their testing would come up all negative until they actually offered the test and given all that was in the Science paper there was no reason to think they would come up that way. They thought they would get plenty of positives.

 

Is it just me or does this whole article and the resulting responses by Cort & Kurt sound more like a Public Relations exercise than anything else? It reminds me of the CAA and their attempts to silence their critics.
I ask the question again to Kurt Rowley:
Will Cooperative Diagnostics be refunding the money they took from patients for a pin-prick test which could not possibly work and which never found a single positive? (one that even Dr Coffin derided at the CFSAC meeting in 2009)

Garcia, you can think what you like, I can't speak for Cort but I am responding because I disagree with that Gerwyn quote. There is no coordination of our responses, in fact i hardly even looked at Cort's post before writing what I wrote.

Will VIP be refunding the money they took from patients if their XMRV testing turns out to produce false positives? What do you think?

These are experimental tests and there is disclosure of that. At least Cooperative took their test off the market when XMRV failed to be confirmed. VIP continues marketing a test that the scientific community doubts is producing real results.

 

Kurt, we've dealt with this before. Here are details of PCR perameters from Satterfield's paper, table two:

What I meant to say was that the CDC/Cooperative PCR study was not based on 62ul of plasma. Gerwyn's comments are misdirected, he tries to convince people that these labs are incredibly stupid, and are practically testing water. Those are not the facts. I don't know the details of how they extract from samples, or even the volume math, but I do understand enough to know that what counts is the PBMCs in the PCR test, not how much plasma is involved in other tests they ran. Again, from the article at the top of this thread:

Specifically, we used PCR to look for XMRV in 2,500 ng of DNA, up to a 25 times larger “haystack” than what was used by Lombardi et al and up to 83 times more than was used by Lo et al. Comparing apples to apples, it was up to 8.3 times more PBMC DNA than used by Lombardi et al.

There is not a huge hole in the skills of these labs, they are PCR experts. And there is not a conspiracy among the labs. You are seeing the scientific facts as they unfold. If Mikovits has more information to clarify what is happening, now would be a good time to publish that, even if it is just a technical report from her lab.

 

Kurt, I'm reeling from your statement "i hardly even looked at Cort's post before writing what I wrote." I thought these comment threads were a discussion. In a discussion one should take on board the prior contributions, otherwise one is just posting propaganda.

You also said "I disagree with that Gerwyn quote." Are you referring to the 62ul of plasma, because I have pasted table 2 from the Satterfield study above, that clearly shows they did use 62ul of plasma.

The difference between VIPDx and CoOpDx is that the VIPDx test gets varied results, both positive and negative, depending on which sample is tested. Of all CoOpDx's samples, they could only get negatives.

You say "the scientific community doubts (VIPDx) is producing real results." I would disagree. Would it not be truer to say that the scientific community is divided in their opinion of the VIPDx test and the HGRV hypothesis at this point in time, bearing in mind we have had four positive studies recently?

 

Kurt, I'm reeling from your statement "i hardly even looked at Cort's post before writing what I wrote." I thought these comment threads were a discussion. In a discussion one should take on board the prior contributions, otherwise one is just posting propaganda.

You also said "I disagree with that Gerwyn quote." Are you referring to the 62ul of plasma, because I have pasted table 2 from the Satterfield study above, that clearly shows they did use 62ul of plasma.

The difference between VIPDx and CoOpDx is that the VIPDx test gets varied results, both positive and negative, depending on which sample is tested. Of all CoOpDx's samples, they could only get negatives.

You say "the scientific community doubts (VIPDx) is producing real results." I would disagree. Would it not be truer to say that the scientific community is divided in their opinion of the VIPDx test and the HGRV hypothesis at this point in time, bearing in mind we have had four positive studies recently?

Reeling? I had already written part of my reply before Cort ever posted, but was busy with other things, and so I did reply without looking over what he wrote. What's the big deal with that? I only mentioned that point because someone said this was a PR campaign, and it was not.

They used 62ul of plasma for one study, but Gerwyn tried to generalize that to say therefore the PBMC level would be impossible to detect, and that is not correct. You have to look at the PBMC levels. I'm not a PCR scientist and neither is Gerwyn, I think people should listen to the researchers when it comes to questions about dilution, so that is why I posted the article info again.

The scientific community is getting less divided over contamination, particularly after CROI. The evidence for some type of contamination event is persuasive to most of the researchers, even Silverman is worried as he knows one possible source. Until the details of a contamination explanation are found there will be many unanswerable questions. I agree that there is still a chance some unknown factor is involved and things may change. But looking at history of past retrovirus hunts this apparently unlikely.

 

It sounds like Cooperative Diagnostics removed their test from the market because it was an absolute failure.
Didn't Silverman just release a new study that found XMRV in the journal Urology?

 

Culturing concentrates viral nucleic acid and is not used for PCR test

Gerwyn did not claim that Lombardi et al cultured prior to PCR (so it makes sense that Racaniello said there's no culturing indicated in the Lombardi et al PCR test).

Lombardi et al cultured PMBCs before they made any attempt to detect viral proteins. It is not the amount of DNA but the concentration of viral nucleic acid that is important. Culturing concentrates the viral nucleic acid- without that step, searching for viral proteins is like looking for a needle in a haystack.

Lombardi found 67% of people to be XMRV+ with gag sequences that they detected using cDNA made from patient RNA.

They also detected HMRV antibodies in ME/CFS patient plasma using cells which expressed the SFFV virus.

What constitutes homology to XMRV?

There actually are no clinically validated positive human samples. That's the whole problem isn't it? Except for the labs in the Science paper no other labs have validated Lombardi's finding and that's how you validate tests.

Although Satterfield agrees with this, Judy Mikovits, Ian Lipkin, Harvey Alter and Frank Ruscetti do not. So we have experienced, award winning virologists at WPI, the Cleveland Clinic, the National Cancer Institute, and the NIH on the one hand, and a newly minted biomedical engineering PhD with a brand new start up company on the other.

Satterfield is certainly entitled to his opinion. And since Satterfield did not want to use the WPIs clinically validated samples, what did he use instead? Heres how he described his choice in his paper:

"XMRV and PMLV are highly related sharing between 87 - 94% nucleotide identity across their genomes and 88 - 97% and 88 - 91% amino acid identity to complete Gag and Env proteins respectively. Indeed, partial Gag (123 aa) and Env (55 aa) sequences from our polytropic HeLa isolate share 96% and 90% identity to XMRV, respectively. Thus, the high amino acid relatedness supports the use of this isolate for [Western Blot] serologic testing."

This was an interesting choice. Satterfield believes that these numbers show that XMRV and PMLV are so highly related that PMLV deserves to be used for his serological assay. However the HMRVs that Lo/Alter found have more in common than the PMLV that Satterfield chose.

By claiming that no one has validated Lombardi et al, he discounted the Alter/Lo work. And yet what Alter/Lo discovered is more homologous to XMRV than the PMLV that Satterfield declares fit for his serological assay. Curious. Bizarre even.

Was there another candidate? Satterfield et al could have used VP62. Lombardi et al. used VP62 for their serological assay and if Satterfield et al had used it, he might have demonstrated that their experimentally constructed antisera could detect these proteins.

Instead, Satterfield is left with an assay that has never been able to detect a HGRV in the blood of a person known to be infected. And since he cant detect it, he declares that it is not there. Ian Lipkin accepts that the WPI samples were valid clinically positive samples and I think we can agree that Lipkin knows a lot more/has more experience than Satterfield.

 

It sounds like Cooperative Diagnostics removed their test from the market because it was an absolute failure.
Didn't Silverman just release a new study that found XMRV in the journal Urology?

Maybe it sounded like that a year ago, but if they did not find a virus that is not present, how would that be a failure? How do you know there is XMRV in CFS patients? Because of a single study that can not be validated by experts?

 

Satterfield is certainly entitled to his opinion. And since Satterfield did not want to use the WPI’s clinically validated samples, what did he use instead?
...
Was there another candidate? Satterfield et al could have used VP62. Lombardi et al. used VP62 for their serological assay and if Satterfield et al had used it, he might have demonstrated that their experimentally constructed antisera could detect these proteins.

Instead, Satterfield is left with an assay that has never been able to detect a HGRV in the blood of a person known to be infected. And since he can’t detect it, he declares that it is not there. Ian Lipkin accepts that the WPI samples were valid clinically positive samples and I think we can agree that Lipkin knows a lot more/has more experience than Satterfield.

WPI does not possess any samples clinically validated in published outside research, you can't validate your own samples. WPI sent samples to the CDC and the CDC tested using more sensitive and advanced tests than WPI and found no XMRV.

Satterfield et al DID use VP62. They calibrated their test to the same virus as WPI, but they used conserved pol regions, which would pick up MORE diversity than the WPI test using gag.

Satterfield to my knowledge has never said they are 100% certain there is no XMRV in CFS patients, there is always a margin of error. He is simply reporting test results and pointing out that in the past these types of conflicting results have always been due to contamination.

 

"Maybe it sounded like that a year ago, but if they did not find a virus that is not present, how would that be a failure? How do you know there is XMRV in CFS patients? Because of a single study that can not be validated by experts?"

How can they release a test on the market intended to find a virus in human blood samples when it has never been shown to be able to that? Where is their proof their test works in humans, when it never did? It rather sounds like they were jumping on the bandwagon to make money, with little concern that the test had been proven to be reliable in humans. Then they withdrew the test, after it was unable to perform.

Don't they have a responsibility to release a test that has shown it can detect XMRV in humans? Where is their ethical responsibility towards the patients?

 

"Maybe it sounded like that a year ago, but if they did not find a virus that is not present, how would that be a failure? How do you know there is XMRV in CFS patients? Because of a single study that can not be validated by experts?"

How can they release a test on the market intended to find a virus in human blood samples when it has never been shown to be able to that? Where is their proof their test works in humans, when it never did? It rather sounds like they were jumping on the bandwagon to make money, with little concern that the test had been proven to be reliable in humans. Then they withdrew the test, after it was unable to perform.

Don't they have a responsibility to release a test that has shown it can detect XMRV in humans? Where is their ethical responsibility towards the patients?

Finding XMRV should not be difficult, based on the Science article. According to WPI the retrovirus can be found using a standard PCR test. Their grad students can find XMRV. I think professional PCR test designers are capable of finding XMRV in a human sample. The problem here is not the competence of the CDC, Cooperative or any of the other labs.

 

Is it just me or does this whole article and the resulting responses by Cort & Kurt sound more like a Public Relations exercise than anything else? It reminds me of the CAA and their attempts to silence their critics.

I do get tired of the idea that there's a kind of "party line" regarding XMRV, and that if anyone even tries to report on anything outside of that party line, they are vilified with ad hominum arguments and accused of being part of a giant anti-patient conspiracy. I, for one, want to hear all sides of the argument. It seems to me that there is enough room for debate and disagreement concerning the results of conflicting studies, without maligning one another's motives.

Kurt, I'm reeling from your statement "i hardly even looked at Cort's post before writing what I wrote." I thought these comment threads were a discussion. In a discussion one should take on board the prior contributions, otherwise one is just posting propaganda.

I didn't know there were rules. I feel free to read and respond to the original article without reading everyone else's opinions first. And sometimes, especially when a thread gets long, I can't get through the whole thing. Given the varying amount of cognitive dysfunction that PR forum members suffer, I don't think we should start dictating standards like that. I think everyone should feel welcome to participate as they are able and willing.

 

For me, this is the most convincing account of why XMRV might be a contaminant that I've read yet.

But there's still so many questions to answer, and it's still too early to dismiss XMRV.

I don't think there's much point in arguing the points between ourselves, because only time will tell us the facts and, reassuringly, there seem to be quite a few studies being carried out by top quality scientists eager to understand the facts (e.g. Lipkin, Singh, Levy, Mikovits, Alter, Lo, the BWG, and a few others.)


Satterfield seems genuine, and is clearly an 'expert' in his specific field.

But the trouble is with 'experts' is that they often stick to rigid rules, and formulaic ways of doing things, and by doing so, can miss things.

Along comes a free thinking individual, and finds stuff that doesn't fit into the experts' predetermined world view, or doesn't fit into expected patterns of behaviour.


There's a couple of points where I thought Satterfield was being particularly stubborn about science needing to fit predetermined patterns.

One was the area of using the cloned virus or the monkey virus to make the PCR. He says that he must do it this way because that's the way it's done within the scientific community. Well, Judy Mikovits is breaking the mold, and maybe that's why she's getting unexpected results.

The other is that Satterfield says the history of contamination shows that this must also be a contaminant. Again, this suggests a certain lack of curiosity, and maybe a case of sticking to the text books too much.

The other thing I noticed is that all of these scientists are saying "this is the way we do science", and XMRV does not fit into our template of how we expect a virus to behave.

There's still a lot of questions to answer, and only further research will answer them.


I think one of the first questions to ask, at this stage is; is XMRV a human virus, or a mouse virus, or does it only exist in cell lines?

I can't help feeling that this virus does not exist only in cell lines, but time will tell.

If it is a human virus, then in which human populations does it reside?


The next question to answer is why does the WPI continue to get consistent and replicable results, no matter which lab they use to test the samples?


I remember, in the early days of XMRV, many of the 'experts' completely dismissed XMRV, and said that it wasn't a real virus, but was just mouse endogenous virus contamination. Now all the 'experts' seem to agree that XMRV is a real virus, and it isn't a mouse endogenous virus but it is something quite unexpected. So who was right all along? Judy Mikovits or all the other 'experts'? Judy was detecting a real virus all along, and not mouse contamination.

They said that Judy had mice in her lab. But she doesn't. It turns out that XMRV is created by a cell line being used in many labs across the world, and yet only Judy can detect XMRV (a real virus). She must be very clever. And she doesn't even use the cell line in her lab.


So, I prefer to keep an open mind about it all, and I tend not to rely on 'experts' too much... I've met too many of them who have let me down personally time and time again.


Also, we need to remember that there might now be more than one source of XMRV. One source might be the cell line. But XMRV might also have jumped into the wild, and infected humans, through vaccines or some other route. It's had many years to do so.


There might be another reason (other than Hue's reasons) why Judy's initial XMRV genetic material is so similar to that of the XMRV in the cell line and to the prostate cancer XMRV. It might be that this was the XMRV type that she detected first, using a very specific test coming from Silverman's study. (But I can't remember all the details on this.) If this was the case, then it would follow that she initially detected and analysed only XMRV very similar to the prostate cancer XMRV, and this would explain why it was more similar to the prostate cancer XMRV than the cell line XMRV, which Hue showed to be the case. Since then Judy says she has detected a wider variety of XMRV's and PMRV's, but has as yet not been able to publish her work, for whatever reason.


We also need to know what Harvey Alter has been doing all this time with his PMRV's.
We haven't heard a peep out of him since he published his study. Why not?

 

We also need to know what Harvey Alter has been doing all this time with his PMRV's.
We haven't heard a peep out of him since he published his study. Why not?

I heard a peep out of Alter on the NIH webcast Demystifying Medicine Chronic Fatigue Syndrome: Is There a Virus? held February 22, 2011.

Both Alter and Lo were inspiring. (Gill was something else.)

You can watch it and look at the slides here.