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False negative results in MLV-related publications

Sam Carter

Guest
Messages
435
It's worth remembering that the prevailing consensus re XMRV/MLVs is not predicated solely on negative PCR studies; rather, it has emerged because the PCR results are in accordance with other lines of evidence such as serological data (Shin 2011), phylogenetic analysis (Katzourakis 2011) and the inability of Mikovits/Ruscetti/Lo to replicate their results when properly blinded (Simmons 2011).
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
Like I said, we are not talking about one or two studies with a small number of samples here. We are talking about (approx.) 15 independent studies (that not all used UNG BTW) involving hundreds of CFS patients' samples.

Even if one lab was stupid enough to change reagent protocol for the experimental testing, it's realistically impossible that most/all labs did this AND that most/all labs did then contaminate all of their experimental samples with UNG or another reagent AND that basically all samples that were contaminated then produced a false negative while all controls consistently produced a positive signal.

Yes, but it might be possible that there are various different factors in all the studies that are causing people to get false negatives.
1. Low copy numbers is the most obvious one, but there are others, as have been discussed over and over:
2. Using inappropriate assays or methodologies.
3. Mixing and confusing contamination with legitimate positive samples.
4. Looking for the wrong virus/variant.
So the use of uracil-DNA-glycosylase is just one factor amongst many.
There are so many unknown factors and variables in this cutting edge research in such a novel field.

At least Lipkin will be using the most cutting edge technology, so I'll hold out for his results.


Although O'Keefe doesn't really mention this, in some studies these controls were actually serially diluted to reflect the reported positivity level and thus should also (at least sometimes) come back as negative (that is, if you have contaminated your samples to such an extent that all of them became false negatives because of it). For instance, in the Switzer et al. study, they had positive controls that consisted of just 3 XMRV copies in 250ng of DNA that were "reliably" positive.

Yes, but what you are overlooking here is that the assay is designed for 100% sensitivity for the spiked virus.
So if a wild virus has less affinity for the assay (i.e. the assay is has less sensitivity for a wild virus), then copy numbers are still an important factor even if the spiked samples have been diluted vastly.


The BWG Phase III (Simmons et al.) findings also speak against this idea. After all, the other labs reported better sensitivity than WPI for the (serially diluted) spiked controls that were mixed in with the patient and negative control samples. This basically shows that their PCR assays were sensitive enough.

But the other labs used different methodologies to the WPI, so their assay sensitivity to XMRV VP-62 isn't entirely relevant. The spiked samples could be irrelevant because we know that VP-62 is very similar to the 22RV1 virus, so if there is a wild virus, then it's likely to be genetically different to VP-62. But I do acknowledge that the BWG study is a major weakness in Judy Mikovits' argument.


It's not "just" me. It's really me and the overwhelming majority of the research community that think the combination of all negative (and other) studies together paint a convincing picture against XMRV being present in those samples "versus" O'Keefe's findings.

Yes, but it would be very naive to believe that the consensus scientific point of view is always the correct one.


If Mikovits/Ruscetti/Lo again cannot reliably discriminate between patients and controls, the search for XMRV in ME/CFS is effectively over (except perhaps for a study that was already in progress). This finding by O'Keefe does nothing to change about that fact, and rightly so.

I agree that there will be no more government funding, but my understanding is that there is more research in the pipeline from various researchers, and rightly so.
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
It's worth remembering that the prevailing consensus re XMRV/MLVs is not predicated solely on negative PCR studies; rather, it has emerged because the PCR results are in accordance with other lines of evidence such as serological data (Shin 2011), phylogenetic analysis (Katzourakis 2011) and the inability of Mikovits/Ruscetti/Lo to replicate their results when properly blinded (Simmons 2011).

I agree Sam. There is a body of (inconclusive) evidence that points towards XMRV being purely a lab contaminant.
However, there is also a body of evidence that says there are many unanswered questions with regards to MLVs and XMRV.
 

barbc56

Senior Member
Messages
3,657
Why on Earth would anyone want a retrovirus to exist??

Only his subjective opinion versus O'Keefe's group saying that it COULD explain the negative findings.

Of course no one wants a retrovirus, Natasa. That's not what I am saying. I am talking about people who are holding on to a cause, because it would explain our illness. We all get frustrated with the lack of research and the psychological modle.We all want to know the cause and that is certainly understandable. What I don't understand is people who are holding on to this theory, looking for any information, however, small or unreasonable and interpreting what researchers are saying through this wish as it would be gratifying to know that a cause has been found.

When the 2009 study was published my hope was that it would pan out but as the follow up studies came in I saw the failings of that study.

I don't think RRM's comments are at all subjective. They are based on the current scientific knowledge that is available at this time. He states the facts and is up front when he gives his opinion. Good for him.

Barb C.:>)
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
I am talking about people who are holding on to a cause, because it would explain our illness. We all get frustrated with the lack of research and the psychological modle.We all want to know the cause and that is certainly understandable. What I don't understand is people who are holding on to this theory, looking for any information, however, small or unreasonable and interpreting what researchers are saying through this wish as it would be gratifying to know that a cause has been found.


Personnally, I'm not convinced that XMRV is associated with ME, and neither do I 'need' it to be the cause of ME, as you suggest some of us do.
I'm following the research out of a keen interest for the unfolding science. Whether, or not, XMRV is associated with ME, the unfolding science fascinates me.

And I don't understand the apparent need by some people on this forum to deny the possibilities before they have been fully explored.

The point that I keep making, is that there are so many unanswered questions about XMRV, and similar viruses, whether they are related to ME or not.
There are loads of questions that have still not been answered... And there is ongoing research... The research isn't finished yet.

I won't list all of the unanswered questions here, because I've already been over them many times.
But there are many...
For example, the issue of antibodies hasn't yet been answered.
And have you read Dr Singh's study? Can you explain its findings with contamination?

Even if the Lipkin study is negative, there will still be ongoing research into both XMRV and MLVs. And rightly so because we don't yet have a full grasp of the MLV science. Copy numbers are so low that it takes cutting edge science to detect them. Novel and unexplained variants and recombinants are popping up everywhere, in many papers. And whether that is as a result of contamination or not, it is still important science. We need to know how many labs and reagents are contaminated for example. And XMRV needs to be contained. What has it contaminated? Has anyone been infected? etc. etc. etc. MMTV keeps on being found in breast cancer samples. Switzer detected very similar viruses to some of the 'VP' variant XMRVs, including an XMRV-Moloney recombinant. This seems like rather a coincidence. Is this coincidence a result of contamination or a wild infection?

So I will continue to explore this subject to the full, as long as it continues to interest me, or until all questions are answered.

And I am open minded about where the research leads, in relation to any of the following: XMRV as a contaminant; XMRV as a cell line virus; XMRV as a potential human virus; MLVs, in general, as contamination; MLVs related to breast cancer (e.g. MMTV); MLVs related to other diseases; MLV/HERV interactions; HERVs that are similar to MLVs as a potential cause of disease; Other HERVs as potential causes of disease; and also the answers to all the questions posed by the existing unrefuted MLV research (some of which is very strong research, such as Dr Singh's.)

If other people are satisfied that XMRV, and related viruses, are a dead-end for ME, and they aren't interested in the field, then that's fine by me.
But if people come onto XMRV threads to try to convince me that XMRV is a dead end, when I'm not yet convinced, or that MLVs shouldn't be studied or have resources wasted on them, then I will vigorously debate.

And I don't understand why people are motivated to post so energetically on the XMRV threads if they don't think XMRV/MLVs are related to ME, and if the subject annoys them.
Why bother? Why not focus attention on areas that you think are more productive areas of research? There are very many interesting areas of research opening up at the moment. For example what they are doing at Bond University in Australia.
Perfectly entitled to post on XMRV threads of course, but I really don't understand, so maybe you could explain barb?
 

barbc56

Senior Member
Messages
3,657
If other people are satisfied that XMRV, and related viruses, are a dead-end for ME, and they aren't interested in the field, then that's fine by me.
But if people come onto XMRV threads to try to convince me that XMRV is a dead end, when I'm not yet convinced, or that MLVs shouldn't be studied or have resources wasted on them, then I will vigorously debate.

And I don't understand why people are motivated to post so energetically on the XMRV threads if they don't think XMRV/MLVs are related to ME, and if the subject annoys them.
Why bother? Why not focus attention on areas that you think are more productive areas of research? There are very many interesting areas of research opening up at the moment. For example what they are doing at Bond University in Australia.
Perfectly entitled to post on XMRV threads of course, but I really don't understand, so maybe you could explain barb?

Unfortunately, our condition attracts a lot of pseudoscience so I have always felt that it is important to speak about this subject. I feel a great disservice has been done to our community in the last few years. A lot of advocacy has ended up backfiring and has caused people to look at our condition in a negative way. Yes, at least more people know about our condition but this all could have been concluded a long time ago. Hopefully we have learned from this. This is why I think it's important for people to speak out just as you feel it is important for you to speak out. I do not see it written anywhere in the rules that you have to believe in XMRV to join a discussion. THAT would be rather condenscending and I am simply expressing my opinions/conclusions based on my interpretation of the science with a little help from reading/listening/learning from researchers/experts and other sources I trust. I do not blindly follow anyone.

My opinions just happen to not agree with some people. They are just opinions. If people disagree that's fine. What other people think about me is not important. Being an advocate is. Being an advocate means not being intimidated to stop posting as I feel this information needs to be out there. After all, it is my health and life involved here. I want my life back. I want to be well again.

If the moderators feel differently, feel that what I post is inappropriate, that's a different story as it's not my forum.

I am science based and will keep on posting to include my two cents worth. There are other people on this forum who are far more knowledgeable about science than I am.

I guess I could ask you the same thing. If you find these topics distressing then why post? How do you get the energy to do this?

I have answered the above questions many times so will not bore people by answering again. You can always look up my posts. I also believe there are unanswered questions but as far as priorities go, I think a different direction is called for.

If we were talking about another topic where there were two studies that ended up not being valid and a lot of other studies which disproved the original, why would we go for the first as the resources for studying our condition are scarce. Again, it's priorities.

I don't have to explain to why I post, but I have. I am not obliged to keep answering questions if they have already been answered or if I feel they are not relevant. I have to conserve my energy for what I think is important. Even then it is sometimes a struggle.

I really think it would help if you looked over the rules of debate and why it is important to look at all sides of an issue. We need to be informed consumers and in the long run this will benefit our community.

I think you underestimate the number of people either on this forum or out in the community who feel as I do.

If you try to analyze me and try to" understand" me, it will drive you crazy.:eek: Let that part go. But keep debating the issues, it can only help our cause, eh?

Take care.

Barb. C.:>)
 

currer

Senior Member
Messages
1,409
Well, as everyone knows I support the retroviral research. I have been an ME advocate for twenty years, and been ill for thirty, so I feel my perspective is valuable. I am convinced that there is immense bias in the way our disease is researched.

My consultant, a very eminent man, who did his utmost to change the way ME was treated and perceived, and was defeated, told me once that "the politics in this field is massive."

"Bad advocacy?" We could not have had better advocacy!
If he could not succeed as a caring, concerned leading consultant, who wanted the best for his patients - and who was prepared to work on our behalf for years, - then the normal course of medicine is being perverted.

So, sorry, it doesnt boil down to straight unbiased scientific research, and I have been around for long enough to understand this.

Most of the posters who understand and support the retroviral research have been driven off this board, by continual negative debate. I think you underestimate the support for retroviral research, Barb, and the damage that perpetual attacks on those of us who want to discuss this research here do to the quality and inclusiveness of this site.

On the political side, I was interested to discover that seven new polyomaviruses have been discovered causing human disease since 2002 and this finding has been accepted without controversy.

http://en.wikipedia.org/wiki/Polyomaviridae
"For nearly 40 years, only two polyomaviruses were known to infect humans. Genome sequencing technologies have recently discovered seven additional human polyomaviruses, including one causing most cases of Merkel cell carcinoma and another associated with transplant-associated dysplasia (TSV), that are natural infections of humans. Discovery of these polyomaviruses and other new--but previously undiscovered--viruses may provide clues to the etiologies for human diseases."

"Polyomaviruses have been extensively studied as tumor viruses in humans and animals, leading to fundamental insights into carcinogenesis, DNA replication and protein processing"

So why are new retroviral discoveries attacked so violently?
Because retroviruses cannot infect humans without a VECTOR, their outer membrane is too FRAGILE, they can only be spread blood to blood, and this implicates HUMAN interventions.

....and then the spectre of AIDS looms up... 25 million dead.....a recombinant of four different SIVs...

What about the MMTV research? So many women die from breast cancer. Why has the MMTV association not been more energetically researched?
http://forums.phoenixrising.me/show...-breast-and-other-cancers&p=243507#post243507

Cancer kills. If they will not research breast cancer and retroviruses, what does that tell you about our chances?
 

currer

Senior Member
Messages
1,409
And Barb, if you are worried about the amount of pseudoscience ME attracts, you ought to realise that this is what always occurs when there is a massive, sustained, and deliberate vacuum of properly funded and planned scientific research.

The problem is not the pseudo science, as you seem to believe, but the deliberate diversion of money and medical research into areas that will never provide an answer to our disease. If the science were not being blocked, the pseudoscience would not exist.

Young people DIE from ME, have been dying for years, and there is STILL no SCIENTIFIC research into it.

Is this because of "bad advocacy" and "pseudoscience?" Please get real !!!!!

There is probably nothing we can do as advocates that can make our situation "worse". It is "worse" and has been kept that way for years. Even family doctors, who have to help their patients with severe ME, realise that the medical "guidelines" they have to adhere to are misleading and unhelpful, and in private have expressed their frustration to me.
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
Unfortunately, our condition attracts a lot of pseudoscience so I have always felt that it is important to speak about this subject. I feel a great disservice has been done to our community in the last few years. A lot of advocacy has ended up backfiring and has caused people to look at our condition in a negative way. Yes, at least more people know about our condition but this all could have been concluded a long time ago. Hopefully we have learned from this. This is why I think it's important for people to speak out just as you feel it is important for you to speak out. I do not see it written anywhere in the rules that you have to believe in XMRV to join a discussion. THAT would be rather condenscending and I am simply expressing my opinions/conclusions based on my interpretation of the science with a little help from reading/listening/learning from researchers/experts and other sources I trust. I do not blindly follow anyone.

My opinions just happen to not agree with some people. They are just opinions. If people disagree that's fine. What other people think about me is not important. Being an advocate is. Being an advocate means not being intimidated to stop posting as I feel this information needs to be out there. After all, it is my health and life involved here. I want my life back. I want to be well again.

If the moderators feel differently, feel that what I post is inappropriate, that's a different story as it's not my forum.

I am science based and will keep on posting to include my two cents worth. There are other people on this forum who are far more knowledgeable about science than I am.

I guess I could ask you the same thing. If you find these topics distressing then why post? How do you get the energy to do this?

I have answered the above questions many times so will not bore people by answering again. You can always look up my posts. I also believe there are unanswered questions but as far as priorities go, I think a different direction is called for.

If we were talking about another topic where there were two studies that ended up not being valid and a lot of other studies which disproved the original, why would we go for the first as the resources for studying our condition are scarce. Again, it's priorities.

I don't have to explain to why I post, but I have. I am not obliged to keep answering questions if they have already been answered or if I feel they are not relevant. I have to conserve my energy for what I think is important. Even then it is sometimes a struggle.

I really think it would help if you looked over the rules of debate and why it is important to look at all sides of an issue. We need to be informed consumers and in the long run this will benefit our community.

I think you underestimate the number of people either on this forum or out in the community who feel as I do.

If you try to analyze me and try to" understand" me, it will drive you crazy.:eek: Let that part go. But keep debating the issues, it can only help our cause, eh?

Take care.

Barb. C.:>)

Thanks for answering my question, barb.
Off course you have every right to post on XMRV threads, as I said in my previous post.
My question was a genuine question, probably born out of frustration, and not an attempt to silence you.
I asked the question because you brought the subject up in the first place - Questioning people's motivations etc.


My feeling is that the fundamental reason why XMRV debates get so heated sometimes, boils down to something you just said:

I feel a great disservice has been done to our community in the last few years. A lot of advocacy has ended up backfiring and has caused people to look at our condition in a negative way.

I can understand why opinion that would lead to frustration.

However, I fundamentally disagree on that conclusion.
I think the opposite is true...

I am of the opinion that the XMRV saga has utterly transformed the world of ME for the better.
I truly believe that there is more hope now, and more progress and funding, as a direct result of XMRV.
My thoughts on this are backed up by people like Suzanne Vernon, of the CAA, who, in one of Cort's latest articles, said the same herself: that XMRV has transformed the field of ME research. And she is not known to be a great fan of XMRV.

So I don't think that XMRV has been a distraction, but I think it has been transformative, and that it has focused minds, brought in funds, and brought in fresh high-profile researchers who would never have got involved with ME otherwise. That's exactly what Suzanne Vernon seems to think as well.

Also, I don't think the XMRV research has been a waste of resources, because it has opened up ME research into immune-related avenues which have been productive, and it has brought different fields together which is also productive. Even if there is no association between ME and XMRV, none of the research has been a waste, because it has led to a deeper understanding of mouse retroviruses and shone a light on the dangers of lab contamination and novel cell-line viruses. It will have advanced the science of retrovirology and the science of viral detection.

Personally, I believe that the XMRV saga has been a totally positive force, and I don't see any negatives whatsoever.

I think that anyone who thinks that ME advocates have done our community a disservice, can't have been aware of what things were like beforehand, just three of four years ago, when Reeves was in charge of the CDC.
I don't think it was possible for people to look at our community or our illness in a more negative way than they did in the past.
We were ignored, dismissed, laughed at and ridiculed, and that has been the case ever since the CDC went to Dr Petterson's practise in the 1980's.
I found the level of contempt to be deeply alarming.
And I've only seen things transformed in the past couple of years. It's almost unrecognisable to how it was when i became ill 8 years ago.

At that time, we had Reeves in control of everything in the USA, and Wessely in control of everything in the UK.
It was an utterly dire situation.
Now the leadership at the CDC has been changed, and Wessely seems to be losing his influence over here...
We have biomedical research funding again, and the diagnostic criteria are being changed at the CDC.
The MRC are taking ME seriously.

OK, so things still aren't good enough, but I just can't believe the transformation that i've seen. I just didn't think it would happen.
The only major blots on the landscape, that comes to mind right now, are the disappearance of Jonathan Kerr, and the publication of the PACE Trial.
But the PACE Trial is a throwback to past practises - I don't think it will ever get repeated now.

Maybe I'm being overly-optimistic again, as I have a habit of doing, and maybe there are other major blots on the landscape.
But it seems to me that things are moving in the right direction, and the landscape has been transformed.

Look at what Dr Petterson is doing, and who he's working with.
Look at Lipkin.
Look at Bond University.
Nancy Klimas.
The Light's.

I wasn't aware of this level of top quality research being carried out before XMRV hit the headlines.

I'm not entirely comfortable with some of the styles of advocacy either. I think some of it has been too vitriolic and unpleasant.
But I firmly believe that it is up to patients to hold the establishment to account, because no one else is doing it.
If that makes the establishment uncomfortable and sit up and take notice of us, then I think that's preferable to being ignored and ridiculed.
I know that some researchers have complained about some patient advocacy tactics, but these patients are only holding people to account, and rightly so in my opinion. If we sat back and did nothing, then Reeves would still be controling our destinies.
 

currer

Senior Member
Messages
1,409
Bob is right, the attempt to portray "XMRV" as negative - whatever the outcome, is unacceptable.

It has directed a vast amount of interest in our direction. I remember Dr Coffin saying he had never heard of CFS prior to his reviewing of the Lombardi paper.

The main outcome for me, apart from my continued interest in the outcome of the Lipkin study, is the realisation that retroviral infection has been associated with a number of human diseases, not only ME.
None of these have yet been accepted. But even if research into "XMRV" or HGRVs is blocked in ME, there will come a time when the weight of evidence for retroviral involvement in other disease is too strong (and too much of a common threat) to ignore.

Then we can expect a reassessment of all the previous disease associations and a rehabilitation of those researchers who were brave enough to state what they believed to be true, in the interests of the patient.
 

SilverbladeTE

Senior Member
Messages
3,043
Location
Somewhere near Glasgow, Scotland
Obviously, the massive effort to clamp down on any research into "new" Human retroviruses is spurred by either/and/or:

greed (not wanting to pay for more hospital cases)
Power (not wanting more "Upsets")
biowarfare research experiments (and yes that is possible, only a fraction of what those lunatic assclowns has come out and that is bad enough)
vaccine contamination
 

RRM

Messages
94
Yes, but it might be possible that there are various different factors in all the studies that are causing people to get false negatives..

For every 'true' explanation of results, there are an infinite amount of 'false' explanations. And like "mixing up" blinded samples is not a realistic partial explanation, this O'Keefe finding is not a realistic partial explanation for these findings.

At least Lipkin will be using the most cutting edge technology, so I'll hold out for his results.
For 'his' HGRV study, he will not really use cutting edge technology. He just collected and distributed samples to labs that said they can reproducibly discriminate between ME/CFS pateints and healthy controls based using 'HGRV assays'.

Yes, but what you are overlooking here is that the assay is designed for 100% sensitivity for the spiked virus.
No, I am not overlooking this. Perhaps you are overlooking the fact that this study also used spiked (mouse DNA) material and not a 'wild virus'?

Assuming your explanation, the results of this study really become null and void. You cannot really assume that the results of a 'spiked' test are generaliziable to wild viruses and then disregard spiked experiments that go against your ideas because they are not 'wild viruses'.

And besides, under your assumption, O'Keefe's suggestion about using internal controls becomes useless anyway. After all, it would then still be equally possible that the internal 'spiked' control would show up as positive (like the external 'spiked' controls are now reliably positive at the level that they would get spiked into the sample anyway), while the 'wild virus' in the sample still could get inhibited.

Yes, but it would be very naive to believe that the consensus scientific point of view is always the correct one.

I agree and I certainly never said anything like that.

I was merely replying to a an earlier authority argument - Natasha said it was me against the O'Keefe lab. That is a bit like saying that, if you think evolution is true, that it is you against professor Behe (implying that this professor will probably know better than you). Of course, when confronted with this argument, it's valid to show that it is not just you against Behe.
 

barbc56

Senior Member
Messages
3,657
Thanks for taking the time explaining your take on the science behind the above questions. It was really helpful and explained some of the questions I have had about this particular research.


Kudos!!

Barb C.:>)
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
Thanks for taking the time explaining your take on the science behind the above questions. It was really helpful and explained some of the questions I have had about this particular research.

I'm glad you understood RRM's last post Barb. I'm afraid I didn't understand anything he said in relation to spiked viruses, so I wonder if maybe you could help me out and explain it to me?
 

natasa778

Senior Member
Messages
1,774
I was merely replying to a an earlier authority argument - Natasha said it was me against the O'Keefe lab.


Argument nothing to do with authority, all to do with pointing to your opinion being just that - a subjective opinion masked as objective words from up high ;)