Reagent Contamination Study

[KFG]

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019953

Apologies if this is posted elsewhere.

 

[Snow Leopard]

They only explicitly question the Lo et al. results.
They conclude with this:

We found that contamination can be sporadic and so not immediately apparent. Although with one manufacturer's kit virtually all water controls gave amplification (Qiagen QuantiTect Virus Kit), the contamination described in this study with IPT reagents only became apparent once a large number of known negative samples (water) were amplified. This contamination of the Taq reagents with murine DNA sequences may be due to the enzyme mix itself and a consequence of the hot start mechanism. IPT has a mouse monoclonal antibody blocking the active site which is heat denatured for activation. This could contain murine DNA, potentially in variable amounts in different lots.

We do not believe that our observations serve to indicate that XMRV/pMLV sequences detected ex vivo in human materials inevitably will have come from the amplification reagents. Nor for that matter can we explain how in some studies there have been very significant differences between the detection rates in cases and comparator groups.

We suggest that contamination may complicate studies of XMRV in multiple ways depending on the source of the adventitious nucleic acid. Presence of material (RNA or DNA) from 22Rv1 cells would result in sequences identical or nearly identical to XMRV. Alternatively, mouse DNA could be the problem in other studies, when a variety of endogenous MLV will be amplified resulting in a range of related sequences as seen in this study. In particular we caution that the reagents used to detect MLV-like sequences may themselves have been contaminated with murine DNA. Although we do not suggest that this is the primary source of the XMRV/pMLV sequences observed in previous studies, it could well represent a confounding factor and note that the original detection of XMRV gag sequences by Urisman and colleagues used Invitrogen Taq and Lo and colleagues used IPT reagents. Finally we believe we would be wise to remember the classical warning Caveat Emptor.

 

[omerbasket]

There are many ways in which a PCR assay could be contaminated - that is well known. The thing is: Both the WPI and the FDA took appropriate measures so that if there would be contamination there, they would know. The WPI included negative controls in every experiment, they checked for mouse mtDNA, and perhaps their most strong evidence is that they have shown antibodies to a gammaretrovirus. Coffin can say all that he wants about these antibodies potential to be because of a friend MLV infection or something like that, but not only that it does not really matter (these are both MLVs, and as I understand it, the treatment would probably be the same) but also that it would be a very strange coincidence that while they were finding XMRV that was due to contamination, they, by chance, found F-MLV or whatever MLV that was really in the person.

The FDA also took appropriate measures to see that it is not contamination: They talk about contamination of the reagents, and that therefore their negative controls came up positive many time. Well, Dr. Lo used hundreds of negative controls, and they never, not even once, came up positive in the assay. They talk about presence of 22Rv1 DNA/RNA? Well, they might even say that this DNA/RNA got into Lo's patient samples while they were stored or processed, and much less to the healthy controls (which would be strange - it did get there, but in much less percentage?) - but how can they explain that when they took fresh samples 15 years later from 8 patients, 7 of them came up positive again? Does the contamination prefer the blood of the sick ones? Not that I'd expect that 22RV1 would contaminte Lo's samples with PMRVs, as it contains XMRV that is almost identical to VP62. Regarding the WPI's samples, again - they found antibodies, which are not produced, to my knowledge, in the lab - and moreover, as Dr. Mikovits said, they checked and as far as one can know, the 22Rv1 cell line (which does not exist in real human beings) was never present in the state of Nevada, ever (and their samples were taken and stored in the state of Nevada).
Next: Mouse DNA? Lo used an mtDNA assay that was extremely sensitive - more sensitive than Coffin's extremely sensitive IAP assay - and he also used Coffin's IAP and improved-IAP assays, and didn't find any mouse DNA contamination. As Dr. Lo showed, his mtDNA assay was 100 times more sensitive than his PCR assay with which he detected the PMRVs, so it makes no sense whatsoever that those were the results of mouse DNA contamination. Regarding the WPI's study, they also used an mtDNA assay on their samples and cell lines and found not contamintion, and again - their findings of antibodies a very, very strong piece of evidence.

So, regarding every suggesment for contamination there is an excellent answer for why it can't be a contamination.

 

[currer]

Stoye has himself been studying how human host restriction factors interact with MLVs .
This was put up on PR a while ago.
http://www.ncbi.nlm.nih.gov/pubmed/21483490

 

[Bob]

Can anyone help me with the conclusions of this study?
I don't understand exactly what they've concluded.
In the conclusion they only say that they found pMLV-like particles, but in the body of the text, they seem to be saying that they found XMRV-like particles.
And the particles that they have found seem to be quite similar to XMRV and PMRV.
Can anyone clarify? (sorry, i'm being lazy - i can't be bothered to print it out and study it carefully!)


I'm wondering where all of these contaminants come from.
Why are they in the reagents in the first place?
And what are they exactly? Is it just mouse contamination?
Are they already known MLV's?
And if they aren't already known, then why aren't they?
I thought that there had been loads of previous work studying mouse retroviruses, so I would have thought that they would have been previously documented.


Questions, questions, questions - as usual!!!

 

[eric_s]

Of course i would prefer another positive study over this, but from the quote i think they have a factual and objective tone and conclusion.

I'm sure the authors of the positive studies are able to verify wheter these things could have interfered with their study's results. Of course it will cost them time...

What i wonder, all this material seems to be "full" with contamination. How can you even sell something like that?? They would have to refund customers, pay for the damages that arose from wrong results etc., i guess. What sort of quality is this? The best the industry can offer? Seems like crap to me...
And there has been tons of research into MLVs before. What material did they use for PCR there? These brands? So what about all this research? Is a part of it in question now, too?

 

[Bob]

What i wonder, all this material seems to be "full" with contamination. How can you even sell something like that?? They would have to refund customers, pay for the damages that arose from wrong results etc., i guess. What sort of quality is this? The best the industry can offer? Seems like crap to me...
And there has been tons of research into MLVs before. What material did they use for PCR there? These brands? So what about all this research? Is a part of it in question now, too?

It is totally ridiculous, isn't it eric!
Does contamination theory now invalidate all previous MLV research?

If it all were to come to nothing then I hope the WPI would be able to sue the manufacturers for all of their millions of research dollars that they've spent.

(I haven't given up hope yet though.)

 

[Jemal]

If these materials are contaminated, other medical materials might be contaminated as well, like vaccinations. Mouse cells are used a lot, unfortunately. And if these reagants are contaminated, their screenings are not good enough.

Let's not forget last year a vaccine was found to be contaminated with a pig virus:
http://children.webmd.com/vaccines/news/20100322/pig-virus-found-in-gsk-rotavirus-vaccine

Nothing to worry about, the pig virus is harmless, really. We just vaccinated 1.000.000 American kids with it.

If such a big pharmaceutical company messes up with a vaccine, everything is possible. We might all be contaminated with a mouse virus, that causes disease in us.

 

[eric_s]

This is possible, but me, personally, i am careful, because it would be such a huge thing that i don't want to say anything that's not proven. But it has to be investigated where the viruses come from, no doubt about that.

 

[Jemal]

This is possible, but me, personally, i am careful, because it would be such a huge thing that i don't want to say anything that's not proven. But it has to be investigated where the viruses come from, no doubt about that.

It's certainly not proven and not even plausible. Just saying it's a possibility.

I got a little worried when a Dutch professor in virology published a hypothesis that XMRV was introduced in humans by vaccinations...

 

[Bob]

If these materials are contaminated, other medical materials might be contaminated as well, like vaccinations. Mouse cells are used a lot, unfortunately. And if these reagants are contaminated, their screenings are not good enough.

Let's not forget last year a vaccine was found to be contaminated with a pig virus:
http://children.webmd.com/vaccines/news/20100322/pig-virus-found-in-gsk-rotavirus-vaccine

Nothing to worry about, the pig virus is harmless, really. We just vaccinated 1.000.000 American kids with it.

If such a big pharmaceutical company messes up with a vaccine, everything is possible. We might all be contaminated with a mouse virus, that causes disease in us.

Yes, I hope they are now going to test all vaccines for contamination from MLV's, based on all of the latest knowledge.

And then test all people who have received vaccines, for infection by MLV's, or MLV-like viruses.

Oh wait, that's exactly what the WPI are doing!!! (and the whole scientific community has turned against them for doing so!)

 

[eric_s]

It's certainly not proven and not even plausible. Just saying it's a possibility.

I got a little worried when a Dutch professor in virology published a hypothesis that XMRV was introduced in humans by vaccinations...

I agree and i hope this work will be followed up on and they get to the bottom of it.

 

[In Vitro Infidelium]

Let's not forget last year a vaccine was found to be contaminated with a pig virus:
http://children.webmd.com/vaccines/news/20100322/pig-virus-found-in-gsk-rotavirus-vaccine

A virus was not found any more than a pig was found in the vaccine - bits of a virus were found. Whether there are health risks from this needs to be explored but some perspective needs to be taken over the presence of miniscule amounts of material that have no known harmful impacts. A child who when visiting a City Farm strokes a pig, will be exposed to massive amounts more porcine DNA than the same child could have conceivably received from the affected rotorvirus vaccine.

The issue of contamination of reagents is fundamentally different to the issue of vaccine contamination - the testing processes involve amplification, the presence of a tiny amount of contaminant can be magnified to indicate a substantial presence, however this will only cause problems if the contaminant is closely matched to the search objective - this is what is being proposed to be the case by Stoye.

IVI

 

[Jemal]

A virus was not found any more than a pig was found in the vaccine - bits of a virus were found. Whether there are health risks from this needs to be explored but some perspective needs to be taken over the presence of miniscule amounts of material that have no known harmful impacts. A child who when visiting a City Farm strokes a pig, will be exposed to massive amounts more porcine DNA than the same child could have conceivably received from the affected rotorvirus vaccine.

The issue of contamination of reagents is fundamentally different to the issue of vaccine contamination - the testing processes involve amplification, the presence of a tiny amount of contaminant can be magnified to indicate a substantial presence, however this will only cause problems if the contaminant is closely matched to the search objective - this is what is being proposed to be the case by Stoye.

IVI

I really hope vaccinations are safe IVI. And as I said I think it's not plausible that viruses are spreading because of vaccinations, but it's not impossible. That a virology professor launches the hypothesis that XMRV could have been introduced in humans because of vaccinations, is a bit worrying, at least for me.

(Also there is a question of perception: in the example of the pig virus I think many parents would rather have their children stroking pigs, then having those children injected with debris from a pig virus - the first may be more unsafe healthwise, but the latter will be very scary to most parents. We don't even know for sure if the pig virus debris is potentially hazardous, who says it doesn't cause problems a decade later? Scientists tend to be arrogant, but in fact there's still much we don't understand about our bodies and immune systems).

We'll see how it all turns out... I think something is wrong though, too many people are now involved with XMRV for it being just contamination.

 

[Bob]

A virus was not found any more than a pig was found in the vaccine - bits of a virus were found. Whether there are health risks from this needs to be explored but some perspective needs to be taken over the presence of miniscule amounts of material that have no known harmful impacts. A child who when visiting a City Farm strokes a pig, will be exposed to massive amounts more porcine DNA than the same child could have conceivably received from the affected rotorvirus vaccine.

Well I think it's slightly different stroking a pig to being injected with porcine viral segments.

The issue of contamination of reagents is fundamentally different to the issue of vaccine contamination

Not really. If many scientific laboratories are said to be contaminated, and it is so difficult to detect the contamination, then surely vaccines are at risk of contamination.

- the testing processes involve amplification, the presence of a tiny amount of contaminant can be magnified to indicate a substantial presence, however this will only cause problems if the contaminant is closely matched to the search objective - this is what is being proposed to be the case by Stoye.

Or if you are injecting children with the viral particles.

 

[toddm1960]

It's certainly not proven and not even plausible. Just saying it's a possibility.

I got a little worried when a Dutch professor in virology published a hypothesis that XMRV was introduced in humans by vaccinations...

Sure would make governments want to cover up any retrovires that are found huh...........

 

[Jemal]

Sure would make governments want to cover up any retrovires that are found huh...........

I am not sure if they would want to cover it up. I don't really believe in the big conspiracy theories.

I do think it's possible that some people and organisations would rather have XMRV vanish. Maybe even some people in the government. If there was ever a panic around vaccines, it could have a huge impact on the public health, as people wouldn't want to have vaccinations anymore.

 

[eric_s]

With politicians i think we also have to see the fact that they are to some degree forced to think in relatively short timeframes. Even though fixing ME/CFS will save money (big time!) in the long run, they want to get reelected. And the next election is in 1,2,3 or 4 years. So even if they see and understand the need, in some way it's against their personal interest to make big investments. Maybe they just hope the sh*bubble bursts with their successor and not with them around... Sorry for the picture...

 

[currer]

Dont forget we have been living with pigs for thousands of years. Pig-stroking is a longestablished human behaviour!
The average medieval knight was more interested in his pigs than in rescuing captive maidens!

I think it is the route of potential infection that presents a new unknown factor. Plus that porcine virus has not come direct from a pig as such, but a cell line. It has been away from the pig for a while and possibly exposed to other factors that may alter it.

 

[currer]

I seem to remember that many recent attempts to link a new virus with disease have foundered on replicability. PCR is so sensitive to tiny perturbations in research methods that it is impossible to finally prove the existance of a virus one way or the other in samples. It is easy to block research if you demand an impossible standard of proof. The best that you can do is to establish liklihood and continue your hypothesis by doing clinical trials, as JM wants to do.

This is how medicine has been done for centiries. So why the outcry when she wants to see if her (willing) patients get better?

At the Invest in ME conference the WPI were confident and happy. JM did not look like a woman with doubts to me. She can take her opponents criticisms' in her stride.