Discussion in 'XMRV Research and Replication Studies' started by Bob, Jun 30, 2011.
But keep your hands where we can see them :tongue:
If anyone reading this thread is wondering why I haven't responded directly to RedRuth's questions re different techniques. The answer is that I do not consider them relevant to the points that I have made, as I hope I have now managed to explain.
Also, my memory for details is like a sieve, and I had to re-read the paper to re-check everything I said in my opening post. I should have done this earlier in the discussion, but I had thought that my opening post was clear enough to explain everything.
Not only did re-reading the paper confirm my initial thoughts, it strengthened my thoughts about the study.
Without any doubt whatsoever, Switzer's discussion and his results are relevent to other negative XMRV studies.
Reading Switzer's discussions re macaque studies, there can't be any doubt that Switzer is talking about the issue of low copy numbers of DNA, RNA and antibodies in the blood. All detection techniques are vulnerable to negative readings due to low copy numbers.
RedRuth says that I am over-interpreting the study. I agree that this one study cannot define the field of XMRV. But if anything, I believe that I have been careful to under-interpret the implications of this study. Switzer did all of the interpreting himself. None of my comments in my opening post are my own interpretation. (Like I said earlier in the thread, it's all in the paper.)
Now, if I were to over-interpret this study, I would say that this study is absolute proof that XMRV is a human virus, and that all existing negative XMRV studies are invalid, due to a lack of knowledge of the behaviour of the virus in the blood. But I didn't say that.
But this study does provide evidence that XMRV is a human virus, and it could provide a reason why all the negative CFS/XMRV studies are inadequate.
Switzer himself states that XMRV is a human virus, based on his evidence.
He also discusses the possible reasons why he can't detect XMRV in the blood:
"Our results are also consistent with those seen recently in macaques experimentally infected with XMRV in which tissues at necropsy are PCR-positive but viremia and detection of provirus in PBMCs disappear quickly, followed by loss of antibody detection , . "
(i.e. due to low copy numbers.)
lol, hands patting back.
This group hug is outrageous!!!
Bob, I wasn't thinking that at all. You've done a wonderful job of keeping track of all the papers and where /what the arguments are.
Redruth, personally I am very happy that you have come back and are looking at this again. Please bear with us and please explain when there are differences of opinion. I hope that this will help your sister one day. We just want to get better and if that means spending our precious and limited energy getting to grips with the science, we'll do it.
Anyway, group hug continues!
But if they don't test blood/PBMCs for provirus how do they know if it's disappeared or not? Given that Lombardi used nested PCR to detect provirus DNA in blood samples, the negative XMRV studies used nested PCR to look for provirus DNA in blood samples but this study didn't use nested PCR to look for provirus DNA in blood samples I honestly don't see the relevance WRT the methodologies.
Another problem is that the main conclusion of this Switzer paper is that XMRV has no role in prostate cancer and this is most likely what explains the pattern of detection of provirus and viremia.
Regarding the original Lomabardi paper I think it's odd that they didn't do some real time qPCR or reverse transcriptase PCR (at least I don't think they did) do they ever explain why?
Why do you think this is odd? Thanks.
Methinks you are not reading my responses to you, RedRuth. Maybe you should re-read the whole Switzer paper. He discusses why low copy numbers of RNA in his study might be relevant for other methodologies, as I have already explained.
Switzer suggests different reasons for the behaviour of the virus in the blood. He does not associate this behaviour with prostate cancer, but he associates it with the macaque studies. Again, you should read the paper. It's all in there.
Why not email the researchers direct and ask the questions. I've found many of them are happy to help and if you explain that you are someone with qualifications who doesn't understand their methods and also explain that you have a sister with ME, then you are more likely to get a reply.
There are usually reasons why people do things in the way that they do. An experienced virologist will know why they act that way and you can only guess as you don't have their experience. It may have something to do with a particular question they were trying to settle.
Why not ask direct and then get permission to share here if relevant after all. Good luck.
It's a good question.
Here is the Science paper addendum which outlines their techniques:
They do say that they have used nested RT-PCR to detect plasma nucleic acid, but that it is the fourth most effective testing technique for them. They seem to prefer using other techniques that involve testing for DNA, cDNA and antibodies, so maybe they concentrate on their most effective techniques. Although they do say that they have tested for RNA, I can't see any results for RT-PCR in the addendum.
I don't really know the answer to your question, and I don't know why they haven't used qPCR.
Mikovits has talked about some researchers' PCR tests being too specific, and that she has calibrated her techniques to be quite unspecific, and then sequences each of her isolates. So I don't know if that's anything to do with not using qPCR.
Just becasue real time qPCR is the standard these days because it will quantify the amount of your target DNA in the sample rather than giving you a simple +/- answer. Maybe if it's a small institute they may not have the equipment to do qPCR.
I've got my off site proxy working now so I've got the original paper and the SOM.
I just noticed that, they say they did some reverse transcriptase PCR for gag sequences but don't show any results. As far as I can tell they also only did the nested PCR for the sequencing not for the data in Fig1.
I'm guessing she means the primer sequences or I suppose the annealing temperature (this makes a big difference to non identical sequence priming), it shouldn't make any difference to qPCR, I would hazard a guess that they just don't have the equipment to do qPCR. Does Mikovits say what she thinnks the discrepancy between her results and the other ME/XMRV results is down to (are all the other ME/XMRV studies negative?) I know I could look this up but I suspect you have the information to hand.
It think it might be interesting if you asked the WPI why they didn't use the techniques you mention.
There were other positive studies that looked in people with ME/CFS. The most prominent one was Lo et al. Dr. Alter (who was an author there) said their study is "highly confirmatory" of the WPI's findings. Other people disagree and say the sequences found are not XMRV, so it's no confirmation.
IrsiCaixa in Barcelona has also found XMRV in ME/CFS. They have presented their work at some conferences (at the 1'st International XMRV Workshop last Sept and at CROI this spring, maybe others), but i don't remember if it was published.
Dr. Hanson also has found MLV-related viruses in ME/CFS and has now expanded her study.
Dr. Bieger in Germany is collaborating with a lab at an university there and they are finding XMRV.
Also VIP Dx (a commercial lab owned by the WPI) and RedLabs in Belgium are finding XMRV, RedLabs have a license for VIP Dx's test.
They say the virus is only present in very small numbers in the blood and that it's a matter of differences in the methods. Maybe other points, i didn't go back and look. Also the cohort selection matters, but if you assume it's present in ~3% of the healthy population, you should find at least some in a large enough cohort.
The WPI has repeatedly stated that PCR testing at current levels of sensitivity, due to low viral counts in plasma, is relatively ineffective.
Most negative studies have elected to only use PCR, despite WPI's warnings, hence they have done nothing more than validate the ineffectiveness of PCR testing at present. They have not disproved evidence of XMRV. They have not progressed the science any further.
Furthermore they have failed to fully utilize the other more effective techniques used by WPI to detect XMRV. Why? Is it because they are not interested in validating XMRV so much as trying to win the race for a cheap commercial assay, or are they merely trying to sabotage the original findings for personal reasons? Either possibility is not such a noble one.
A simple search on this forum will reveal that many of those involved in negative studies have patents on XMRV strains, representing conflicts of interest (arguably fraudulent misrepresentation). Other groups who have wasted millions of dollars of research funding on dead end investigations also have much to lose in future funding. If anyone still doesn't understand the high stakes involved and that egos and greed are involved, then they need to do a lot more reading. XMRV, if proven to be the cause of me/cfs will destroy the careers of many, from researchers to bureaucrats to patient advocate organizations.
Contamination and recombination will ultimately be proven to be integral elements in the genesis and spread of XMRV and other variants, but not in the way the contamination cabal has tried to portray. Ironically there is a kernal of truth amidst the lies. Switzer's latest study and the report of rampant contamination and high rates of transmission of MLVs in exnografts is the beginning of the end for the contamination lobby, but should also send alarm bells ringing.
MLV recombinants were created in the lab and carelessly released into the public arena. Those responsible must be held to account. Those who are callously covering up this culpability must also be held to account, because every minute another life is lost, and the suffering of millions continues.
Furthermore, I don't believe we have begun to plumb the depths of MLV recombinations yet. While XMRV shows low propensity for mutation, MLVs appear to have high recombinant propensity, with individual strains displaying unique pathogenesis. Who knows what forms will eventually arise? Potentially it could be more devastating than HIV.
The WPI kindly did a table showing discrepencies in method by the various other XMRV studies
There is a link to it here on this website
Mikovits and Lombardi have done so many interviews, spoken at conferences and fielded questions on their methods. They may have already answered your questions. There are been Retrovirology conferences since XMRV was discovered and the researchers have had a chance at close contact to ask the WPI questions and to swap hints/problems. If you want to know about qPCR, then they are sure to have asked the WPI at a conference or similar.
This is a bit like a long conversation. Most of us started at Day one when the paper was published and have followed each development. When someone new comes into the conversation, they may find that the bits they want to talk about have already been covered. It's not as if anyone is going to say "ah ha" and find anything deeply hidden or significent as it's been so well thought through and we've had the chance to email the actual scientists and ask any questions for a very long time.
It's hard for patients to keep going back and find bits for others. As you are a "well person" I don't think it it too much for us to ask you to please do as much research as you can.
The BWG is working out the different methods and may be some hints for you here
Thanks ukxmrv. You explained what I was feeling in a much more restrained fashion. I am too tired to dig up references and go back over the same old ground. Credit to you and the others (Bob) for your patience in this thread. I hope I haven't been too strident. If so I apologise to all.
Yes, Rusty! You said it exactly right. I hope the truth wins out soon and the egos have to bend to the facts. It's time to think of the massive suffering and get to work on the alleviation for all those who have been ignored and discounted.
If it's true that they don't recommend using PCR to screen for XMRV then it's not really an issue, though PCR is one of the methods they used in the original Science paper. I'm just trying to understand what the furore over the methods is given that PCR is generally a very robust technique.
Thanks, that's quite a lot of reading up and cross checking. I only asked Bob because I assumed he would have the information to hand and I wouldn't know where to look.
You can also try a Google Site Search
Separate names with a comma.