Saturday, January 1, 2011

A MUST READ if you are XMRV positive

By Gerwyn:

"I have been informed that Dr dusty Miller plans to use the controversial IAP assay in his forthcoming study to test for mouse contamination instead of the established and much more specific assay for mouse mitochondrial DNA.

I am not in a position to confirm that this is true

My concern lies on his plan to use IAP to detect mouse contamination. This is claimed to only amplify mouse DNA by Coffin


these sequences are found on activated T cells




Anonymous said...

This is false. There are no IAP's in the human genome. Try search on gene bank for the sequences and see for yourself. Furthermore the number of copies of an IAP in the mouse genome is far more than any one of the mitochondrial genes.

The IAP is the best and most reliable to use for mouse contamination. I should know I do lots of PCR in my work.

adustymiller said...

Please tell us what makes you think that the mouse IAP assay can also amplify sequences present in human DNA. The references you cite don't speak to this issue.

One way to test your hypothesis is to take the IAP primers used in the recent Retrovirology papers and check for related sequences in the mouse and human genomes. The sequences are:




When I do this analysis by BLAST search of the NCBI databases, I find many sequences in the mouse genome that show 100% identity with either sequence, but none in the human genome. Thus, both sequences are highly mouse-specific.

In addition, data presented in one of the recent Retrovirology papers (Oakes et al., Fig. 5) shows no amplification of specific IAP bands in human DNA using these primers, but clear amplification of very small amounts of mouse DNA, in contrast to what you claim.

Please enlighten us regarding your claim.

Anonymous said...

If it only tests positive for XMRV, then maybe they can use it as a biological amplifier for XMRV. They're still actively looking for a fast and high throughput method to detect XMRV.
Maybe they found it in the opposite place. Haha.

Cort said...

Most of us (including me) are out of our depths here and we have to decide who to trust on these issues. Gerwyn has CFS and, of course, is rightfully very concerned that these studies be done correctly.

I would note that Dr. John Coffin, Dr. Dusty Miller, Dr Racaniello, Dr. Rein and Dr. Dove have all recommended using IAP in preference of mtDNA. Several of these retrovirologists have well over 100 publications to their name.

I will go with them and I look forward to Gerwyns comments.

KaB said...

As we all know how Cort handles information it's interesting he wants to get involved in this discussion.

He may say that there are some who claim this is the best assay to pick up contamination, but other big names do disagree, and several of those have well of 100 publications to their name. So this bit only distracts everyone from the issue. Then there is the person claiming to be Dusty Miller, we don't know if they are. So we have to wait for official communication.

Finally, we don't have to take anyones word for it at all. Scientists must provide evidence in their research that it is not capable of picking up human IAPs. Once published other researchers will assess if anything is missing or flawed, as is the normal process.

GD said...

As we all know how Cort handles information it's interesting he wants to get involved in this discussion.

He may say that there are some who claim this is the best assay to pick up contamination, but other big names do disagree, and several of those have well of 100 publications to their name. So this bit only distracts everyone from the issue. Then there is the person claiming to be Dusty Miller, we don't know if they are. So we have to wait for official communication.

Finally, we don't have to take anyones word for it at all. Scientists must provide evidence in their research that it is not capable of picking up human IAPs. Once published other researchers will assess if anything is missing or flawed, as is the normal process.

RRM said...

This Gerwyn argument makes no sense at all to me, even beyond the comments by Miller.

If the IAP assay would pick up human sequences, that would still only be a problem (in this context) if Miller would test the actual WPI samples used in the Science study, instead of freshly collected samples from WPI patients. How on earth could Miller falsely "demonstrate" that WPI contaminated their samples by finding IAP positives in samples that he himself collected?

If Gerwyn's argument had any merit to it (which I guess isn't the case given Miller's comments), he (or she) should warn the WPI and FDA/Lo about retesting their old samples using the IAP assay, and not other researchers about testing freshly collected WPI samples.

Independent labs finding IAP positives in just WPI patients and not in controls, would clearly be a good thing for Gerwyn's case as long as the independent lab collected and handled all (blinded) WPI and control samples in the same manner.

Jo said...

RRM: There is a guy going around claiming that Miller is going to use WPI samples. That is why Gerwyn has posted this. Also, we have no idea if the person calling themselves Miller here, is THE Dusty Miller. And people are talking to other researchers involved in MLV-related retroviruses about this IAP test.

RRM said...


The point is that Miller will not be using the actual positve WPI samples but freshly collected samples from WPI patients. If he were to use the actual old samples, he would not be recruiting patients but would have asked the WPI about testing their stored samples.

With that in mind, Gerwyn's comments appear beyond silly. Or perhaps there is another point to his/her critique which I, with my limited intelligence, am apparently missing.

Jo said...

It doesn't matter if the samples are new, if the assay can potentially pick up human IAPs. This needs to be ruled out first. Therefore it is no Gerwyn's comments that are silly.

RRM said...


Sure it is silly. If an independent researcher collects fresh blood from the WPI 'Science' cohort and finds IAP positives in the samples deemded to be positive by Mikovits, it would strengthen the case of Mikovits instead of weaken it. Any test that could differentiate between the WPI positives and healthy controls would.

Say an independent researcher collected blood from 10 XMRV+ 'Science' patients and 10 healthy controls. Can you give me just one example of a possible result that could possibly 'bury' the Science findings by using the IAP assay, even if we assume the assay could 'potentially pick up human IAPs'?

I'll tell you: you cannot, and that is why Gerwyn's argument is indeed beyond silly.

Leave science to the scientists and not to quasi intellectual internet forum warriors.

adustymiller said...

"adustymiller" is indeed Dr. A. Dusty Miller. I have a lab at the Fred Hutchinson Cancer Research Center.

Given all of the controversy surrounding the claims that XMRV and/or related retroviruses are present in humans, my lab members and I want to test for the presence of XMRV in samples freshly obtained from those most likely to carry the virus. This includes those who have tested positive by PCR and/or virus assays performed by other labs. However, we have not currently set up formal arrangements with other labs, including the WPI, to obtain samples from people they have tested. Instead, we plan to rely on volunteers from the CFS/ME community to provide small samples of blood for analysis.

I have been in communication with Ecoclimber and Cort to refine the objectives and criteria for our study and to help with recruitment of subjects. We understand that there is heated controversy over how the study should be done, but I hope my extensive experience in retrovirology provides some assurance that we can perform this study properly.

Regarding the IAP issue, published results and our analysis indicate that PCR assay for mouse IAP sequences provides the best method to rule out mouse DNA contamination in our study. One can find relatives of these sequences in the human genome, but they are not closely related and do not amplify with the published IAP primers.

I want to assure the CFS/ME community that I have no hidden agenda, other than that I would like to apply our expertise in retrovirology to contribute to an understanding and possible treatment of CFS/ME. This predisposes me to want to find the virus in humans, but I clearly understand that false positive results are not in anyone's best interests.

Please pardon me if I can't address many questions raised on the various blogs. I already spend way too much time in the lab!

Anonymous said...

If you are really are Dusty Miller, you will also know that if you prodused false negatives that would also be a devastating big problem.

We have not yet seen your research designed, and therefore its not easy to find our if you research will bring positive effects for us ill with me/cfs.

And also if you are Dusty Miller and are of a need for xmrv positive too you research, why not contact the WPI and they can contact us. We trust the WPI.

Jo said...

This is ridiculous. I know who Dusty Miller is. But unless you choose to communicate to people through official means, there is no way anyone can believe you are Dusty Miller. Anyone could do that.

What kind of researcher would talk to Cort Johnson about how to design a study? I have no idea about Ecoclimber, but again, and you serious??? You talk to those doctors and researchers who are experts in the biology of the disease.

RRM: How would finding IAPs in those already testing positive make the case stronger for MLV-related retroviruses? Do you even understand how this works? This assay is to test for contamination.

Anonymous said...

No, I'm spartacus. So which is the real Dr Miller? ha ha ha

adustymiller said...

In response to Anonymous January 3, 2011 9:24 AM:

A basic tenet of science is that results obtained in one lab should be reproducible in other labs. We plan to use many of the same techniques used in the Lombardi et al. study from the WPI, and some additional techniques we have developed, to detect XMRV. One of the criteria for inclusion in our study is that subjects must have tested positive for XMRV by another lab. Thus, we hope to include subjects who are likely to be positive, and it would be very helpful to study some who have previously tested XMRV-positive by the WPI.

If we find positives, we will have reproduced the findings of the WPI and others, and will continue to work on the virus and its possible role in CFS/ME. If not, we have more work to do to figure out the cause of the differences between our results and the positive results of others.

As you know, there is much current controversy over whether XMRV is present in humans or not. I will do my best to avoid adding to the confusion and to help resolve the issue.

Anonymous said...

sequence similarity between rodent Iap s and HERV-k assay very likely to provide false positives for mouse contamination

Anonymous said...

If Miller uses a novel unproven assay, that has not been shown capable of detecting real live MLV-related retroviruses, and all he gets is IAPs from his not proven to also detect human IAPs. That's it. End Game. Whether MLV-related retroviruses are there or not. And no evidence right now exists to show they are not there.

Anonymous said...

Thanks Dr Miller!

Anonymous said...

More from Gerwyn at

Ok the details so far PCR only no controls(so you cant tell if the IAP assay is amplifying human DNA or not) undisclosed primers no plans to amplify.

PCR is up to 400% less sensitive than other assay methods . The chance of detecting it without amplification are slim

IHC ,FISH and IFC are much more sensitive

To cap it all he is going to use IAP This methodology is straight out of the Mclure handbook. This is even worse because this guy knows mulvs!!

this has all the hallmarks to a study designed to find contamination or not find xmrv


Gerwyn said...

Dr Miller is not planning to replicate the lombardi methodology in any way. Contrary to uninformed opinion earlier there are IAP sequences in the human genome which have sequence homology with the mouse IAP pol gene and herve K sequences.Thus this assay has the theoretical capability to amplify human DNA.I would also like to remind XMRV positive people that no one who has not used the Lombardi et al methodology has ever detected XMRV.Do they want another false negative PCR study on their hands.This could be fatal there is only one way a scientist would advocate testing a hypothesis and that is experimentally. Any one who suggests alternative methods is highly unlikely to be a scientist or speak for one!

RRM said...


Sure I understand how the IAP assay works. That's exactly my point: how can a contamination assay cast doubt on samples that were not collected by the lab in question?

If the IAP assay returns positives on Miller's 'Science' samples, it would only imply that HE had contaminated the freshly collected samples, and would not cast any (more) doubt on the WPI's 'Science' results.

Only an IAP test on the old 'Science' samples, the ones that were actually used, could potentially hurt those old results.

ME/CFS commenting said...

If this study does proceed, it needs a good number of non-infected controls put in place, according to Rein. He expects about a 10% false positive rate.
Might be a good idea to confer with WPI, too. Hmm, and what do Alter and Lo have to say about this in preference to their mtDNA?

Dr Speedy said...

Sequences etc are fine, but contamination can NOT cause an antibody response or infect other human cell lines

Anonymous said...

It depends on if the IAP assay works. If it is not proven to not also detect human IAPs thats a problem. Once that is settled great. The next issue is will Miller be using multiple methods. To not do so is foolish.

Alter disagrees about this assay, he says the assay he used is more sensitive. He said this at the recent FDA blood meeting.

Dr Speedy said...

Professor Racaniello: Why XMRV is not a laboratory contaminant

Anonymous said...

Gerwyn is also a scientist - a fact that Cort, as usual, conveniently left out.

Dr. Miller, anything that has Cort's fingerprints on it will be suspect for many ME/CFS patients. He has ground away at the work and reputations of WPI and Dr Mikovits since the Science publication. You just can't trust him.

Of course, scientists may disagree. That is the nature of science. But we also know that there is a great deal of politics and economic selfishness involved in the attempt to delay, sabotage or squelch ME/CFS virology research.

Anonymous said...

The most prudent thing for Dusty Miller is to recruit non tested well defined ME/CFS sick. If he use Canada Concensus criteria he will find at least 67 % and higer positives in this group, and it will not lead to more problems for him.

And this would not be so damanging to us if he can not find XMRV with his testing metodes. It would just be another negative study.

The real harm us is if he tests xmrv tested positives from WPI, and he can not find XRMV. And then he publishes that the WPIs positives where false positives.

That is so dangerous.

Anonymous said...

This is why the Miller study will be negative and shown to be contaminated.

Miller is going to use VP62 synthetic clone of XMRV and the 22rv1 prostate cancer cell line. Water or blood spiked with VP62 or 22rv1 should in theory pick up these viruses. And it is also reasonable to assume that if you add primers to detect more variable sequences, then you have a good chance of picking up more strains/viruses. The evidence however does not support this position, other factors seem to come into play when attempting to detect MLV-related viruses in a persons blood. As all studies that have used VP62 and 22rv1 have been negative.

Switzer et al. used the Lombardi primers, but the sensitivity of their assay was ascertained by calibration with VP62, but this PCR assay could not detect XMRV in known clinical positives sent by the WPI or detect population levels determined by 4 independent groups of workers

Groom et al. and van kuppelveld et al. using the same approach also failed to detect even population levels of XMRV.

To cap it all, the Blood XMRV working group has experienced the same problem when assays proved sensitive in vitro (not in a living organism) have failed to find XMRV in known positive samples

The IAP assay for checking for mouse contamination is also flawed, as humans produce IAPs, and there is the possibility of mouse IAPs transferring to humans by mouse viruses.

Therefore this methodology is a failed one, and as I said the study will be negative and proven as contamination even though the IAP assay is flawed. And worse it will be so for WPI positive patients.

jace said...

As my wise mother used to say, "When in doubt, don't"

I find it strange that Dusty and Cort are trying so hard to get people tested positive by WPI to partake in these studies, and I find their attempts to denigrate others disgusting.

Methinks they do protest too much, to paraphrase Shakespeare.


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