• Welcome to Phoenix Rising!

    Created in 2008, Phoenix Rising is the largest and oldest forum dedicated to furthering the understanding of and finding treatments for complex chronic illnesses such as chronic fatigue syndrome (ME/CFS), fibromyalgia (FM), long COVID, postural orthostatic tachycardia syndrome (POTS), mast cell activation syndrome (MCAS), and allied diseases.

    To register, simply click the Register button at the top right.

Dr Singh Talks on her CFS XMRV study and the WPI's Response

Cort

Phoenix Rising Founder
The Human Isolate Question-The WPI appears to propose that their use of a human isolate of XMRV allowed them to find an XMRV-like family of viruses containing VP62 strains, p variants of the virus (Lo et. als pMLVs) and other strains and that Dr. Singhs use of an XMRV clone developed from the VP62 strain lead her to miss those strains and that is one reason that she didnt find anything.


(Note though that the WPI did say that the patients they directed to Dr. Singh tested positive for antibodies to the VP62 strain - which, given the increased sensitivity of Dr. Singhs tests she should have been more effective at picking up.The WPIs argument regarding using a human isolate possibly explain reduced results but its hard to understand how it could explain zero results. The data from the original study indicated that tests using VP62 were adequate to pick the type of XMRV found in that study. ).


I asked Dr. Singh if she felt her use of the VP62 clone impaired her ability to find all the XMRV or XMRV-like sequences that were present? She replied that retroviral tests should be able to easily pick up the amount of variation that has been published thus far.


VP62 is a human isolate of XMRV - it was isolated from a patient with prostate cancer. There are hardly any differences between VP62 and XMRV isolates from the Mikovits lab - a total of 8 to 32 bases among 8000 bases. That is a difference of only 0.1 to 0.4% - which for retroviruses is not much variation at all, given that they utilize an enzyme that cannot proof-read for their replication. Using any one of the 6 published isolates of XMRV should lead to outcomes that are not significantly different.

Exact Replication? Dr. Singh reported in her paper that she replicated the methods of the Lombardi paper but there were differences. I asked if she was could be getting different results because of small differences in preparation and testing?


She stated


We used a total of 9-12 assays on each sample to look for XMRV. Some were new assays that we developed - and these assays are far more sensitive than any of the assays where sensitivity data is shown. Others were a replication of assays performed in Lombardi et al or Lo et al. The viral culture was done exactly as explained by Dr. Ruscetti.


And then explained one difference that she felt was missing in both the Lombardi and Lo studies which meant the studies were prone to contamination; ie there was one part of those studies that she felt compelled not to replicate.


The nested PCR was done as described in Lo et al, with one small modification. We used the dUTP-UNG system to prevent contamination of our lab with PCR products.

Since nested PCRs involve opening of tubes containing a PCR product, the danger of this product contaminating other equipment, reagents and samples in the lab is very real. Once contamination has occurred, it is very difficult to get rid of it. Any reputable lab that uses PCR as a test on clinical samples, uses dUTP-UNG. It has been shown to not affect the PCR itself, and to provide a very real safeguard against contamination.

I do not know why Lo et al or Lombardi et al used a process prone to contamination like the nested PCR, and then did not use the dUTP-UNG system (you'll have to ask them). But I could not risk contaminating my lab with PCR amplicons - then every subsequent test we did would be suspect.
 

Cort

Phoenix Rising Founder
Dr. Satterfield of Cooperative Diagnostics on Dr. Singhs XMRV CFS Statement

Dr. Satterfield of Cooperative Diagnostics explains about the dUTP_UNG system and provides his take on Dr. Singhs comments on XMRV..

Dr. Singh - The nested PCR was done as described in Lo et al, with one small modification. We used the dUTP-UNG system to prevent contamination of our lab with PCR products.

Dr. Singh is right adding the dUTP-UNG system is a good idea for any non real-time PCR, and especially for multistep PCRs like the nested PCR. UNG does not have a negative effect on the PCR but does reduce the risk of contamination a LOT. While I was at Arcxis, we ran into a number of labs that would not buy PCR tests unless they had UNG present. It had become a standard in US clinical practice. That being said, basic research often does not use UNG.


The basic principle is as follows. Each PCR produces around 100 billion copies of the amplified product. The PCR reaction takes place inside of a fluid in a plastic tube. In order to analyze the reaction (unless you are using real-time PCR), you have to open the top of the tube to get access to the fluid. The top often pops open, releasing a microscopic spray into the air, kind of like when you sneeze. This spray potentially contains billions of copies of DNA that can contaminate other PCR reactions.

It will not contaminate culture and it cannot contaminate antibody tests. But it will contaminate other PCR tests using the same or similar primers (ie looking at the same gene). As Dr. Singh said, once this type of contamination is in a lab, it is almost impossible to get rid of short of burning down the entire establishment.

So how does the dUTP-UNG system help safe guard against this type of contamination? UNG is an enzyme, a protein that performs a function. If you recall from biology class, there are four standard DNA bases: A, C, G, and T. The dUTP-UNG system substitutes the T for an RNA base, U (ie this type of PCR uses A, C, G and U instead of the normal four). At the end of the PCR, instead of having a true DNA product, you have a DNA product interspersed with the RNA base, U. The UNG is activated at the beginning of the PCR reaction. It recognizes the U base in the DNA product and destroys the DNA products with U bases present.

That means, even if you had contamination in the lab from a previous PCR reaction, the UNG will provide a lot of protection against this type of false positive. We played with UNG to try to quantify exactly how much protection it provides. We found it would prevent false positives if less than around 1,000,000 contaminating copies were present in the tube. That is a large number, meaning that it should prevent contaminationfrom prior PCR reactions in almost every case.

Someone called us to ask if we used UNG in our reactions. We do not. But we do not use a PCR that has to open the top risking contamination of the lab. The easiest way to prevent contamination in real-time PCR is to simply never open the top of the tube after the PCR is finished!

Dr. Singh- "Since nested PCRs involve opening of tubes containing a PCR product, the danger of this product contaminating other equipment, reagents and samples in the lab is very real. Once contamination has occurred, it is very difficult to get rid of it. Any reputable lab that uses PCR as a test on clinical samples, uses dUTP-UNG. It has been shown to not affect the PCR itself, and to provide a very real safeguard against contamination.

I do not know why Lo et al or Lombardi et al used a process prone to contamination like the nested PCR, and then did not use the dUTP-UNG system (youll have to ask them). But I could not risk contaminating my lab with PCR amplicons then every subsequent test we did would be suspect."

Lombardi et al clearly ran their PCR as a research test in the Science article. UNG would not necessarily be expected in a first paper. Their use of culture and antibody testing would have been done to address any risk of false positives arising from PCR carry over contamination.


But now with all the WPI data in question, the use of UNG in a nested PCR would be very important. Nested PCR, even more than single round PCR, is subject to contamination. It has a first round of PCR potentially creating billion
s of copies of DNA from as little as one starting copy. Then the top is opened (remember the sneeze effect) and some of the fluid containing those billion copies is placed into a second PCR to create even more copies. The second round of PCR compensates for any inadequacies in the design of the first round PCR. Then the top of that reaction is opened (a second sneeze) to analyze its contents.


While this clearly will not explain the antibody positives or culture positives, it would be a good practice to implement in this type of reaction. Of course, even the addition of UNG will not prevent other types of contamination, such as the presence of virus in the polymerases being used, or the presence of virus on lab equipment, etc.
 

Megan

Senior Member
Messages
233
Location
Australia
Cort,

Thanks for your interview. I accept that there is weighty evidence is against XMRV now, but it still seems to me there are outstanding questions regarding both XMRV and other possible virusus/retroviruses. One of these is in relation to what were the positive antibody tests in referred to in Singh's patent document picking up? The relevant quotes from the patent are below. I understand both of these tests to be antibody tests (ie not subject to contamination). I suppose she would say they are cross reacting to something else, but the question remains what? especially if the tests are negative in controls as stated. Surely this is an important question that we CFS patients ought not let go?


Quotes from patent document:

[0027] Figure 13 shows Western blot detecting presence of antibodies against XMRV polypeptides. A serum sample from a patient with chronic fatigue syndrome shows antibodies against SU, CA and pl5E polypeptides (A). In (B) is a negative control serum, which does not show reactivity to any of the XMRV polypeptide.

[0134] Similarly, to detect if patients with Chronic Fatigue Syndrome have evidence of XMRV infection, we collected sera from 105 patients diagnosed as having chronic fatigue syndrome and fulfilling the Fukuda criteria for diagnosis. For comparison, we also collected sera from 200 healthy volunteers. Each of these sera was used to probe a PVDF membrane onto which XMRV proteins had been transferred from a gel. A positive sample usually contained antibodies reactive to at least two of the following three XMRV polypeptides: SU, CA and pi 5E (see Fig. 13A). Non-reactive samples (most healthy volunteers) did not contain antibodies to SU or pl5E (Fig. 13B). Antibodies to CA alone, however, were quite prevalent in the healthy volunteer population, and are not to be interpreted as evidence of XMRV infection.
 
Messages
118
What's this look who's presenting at Cold Spring Harbor Retrovirolgy conference on May 23, 2011. I guess she did find it in prostate cancer with a different assay.

Singh, I.R. XMRV, or a related virus, is present in a large percentage of men and is localized to the androgen secreting Leydig cells of the testis
 

August59

Daughters High School Graduation
Messages
1,617
Location
Upstate SC, USA
What's this look who's presenting at Cold Spring Harbor Retrovirolgy conference on May 23, 2011. I guess she did find it in prostate cancer with a different assay.

Singh, I.R. XMRV, or a related virus, is present in a large percentage of men and is localized to the androgen secreting Leydig cells of the testis

I wonder if she will pull her poster now? Cozak, Sarafianos and Paprotka will be represented at this conference as well by either a poster or talk. Could be interesting!

http://meetings.cshl.edu/meetings/retro11.shtml
 

eric_s

Senior Member
Messages
1,925
Location
Switzerland/Spain (Valencia)
Will be interesting to see what method she used to find XMRV. I guess she looked somewhere else than in the blood, as she found nothing there in the ME/CFS study, but if she says it's found in a large percentage of men, it can't be prostate tissue either, i guess.

A large percentage sounds like something higher than ~4%. Interesting... maybe something we've never heard before.

Edit: Oh, ok, the text probably answers my question already. But what lead her to look there? Maybe her autopsy study?
 

Cort

Phoenix Rising Founder
That's an amazing statement...XMRV or a 'related virus' and its in the testes....that's got to be the autopsy studies....

One question is is it doing anything? If it's the autopsy study she should be able to determine if XMRV's presence is associated with illnesses. If nobody had any physical problems that would seem to indicate testical problems then she will probably scratch XMRV off the 'damage forming pathogen' list.

It would be disappointing if that was the only place she found it..

Of course that statement does indicate that XMRV is a virus that infects humans - which she has always believed.

It could end up working out both ways....XMRV has escaped from the lab and contaminated samples via the 22RV1 cell line and it is a virus that is, out in the wild so to speak, and is infecting humans. Its even possible that the WPI has examples of both! That the 22RV1 cell did get into Silverman's samples and hence to some of the WPI samples and they also picked up the wild virus as well in their sampling. :)

Silverman's paper will be so key....has he been able to uncover contamination or has he been able to discount that. Can he now prove that his XMRV integration sites reflect human infection and not contamination (as an earlier paper suggested) or do they reflect contamination?

XMRV, or a related virus, is present in a large percentage of men and is localized to the androgen secreting Leydig cells of the testis
 

Gemini

Senior Member
Messages
1,176
Location
East Coast USA
That's an amazing statement...XMRV or a 'related virus' and its in the testes....that's got to be the autopsy studies....

Both her blood and tissue results are consistent with Emory's macaque findings
where XMRV disappeared from blood rather quickly and was later found in organs...
 

August59

Daughters High School Graduation
Messages
1,617
Location
Upstate SC, USA
The website for the conference states that all of the abstracts had to be submitted by March the 8th. That is a few weeks back! That is why I was asking about her pulling it, but I imagine her study was already completed by then anyway. Dr. Cosak has been very quite lately too, so I curious to see what she brings to the conference.
 

Cort

Phoenix Rising Founder
It could end up working out both ways....XMRV has escaped from the lab and contaminated samples via the 22RV1 cell line and it is a virus that is, out in the wild so to speak, and is infecting humans. Its even possible that the WPI has examples of both! That the 22RV1 cell did get into Silverman's samples and hence to some of the WPI samples and they also picked up the wild virus as well in their sampling. :)

Silverman's paper will be so key....has he been able to uncover contamination or has he been able to discount that. Can he now prove that his XMRV integration sites reflect human infection and not contamination (as an earlier paper suggested) or do they reflect contamination?

I think my statement was misinterpretated by someone to suggest that I believe this finding rescues XMRV from the plight it is in with CFS. I don't. I think XMRV's connection to CFS rests in labs finding it in the blood of people with CFS - not in their tissues. My personal opinion is that - after a year and a half of searching for it - that that's not going to happen. The most likely scenario IMHO is that Silverman will announce at the conference that the 22RV1 cell line contaminated his prostate samples thus probably providing a smoking gun pointing at the WPI.

On the other hand Silverman could say that the opposite; that his prostate findings are real and that 22RV1 did not contaminate his samples. Since there is new evidence pointing to XMRV being an genetically distinct infectious virus - I think its possible that Dr. Mikovits picked up some of that if she is using tests that search for an array of viruses. She did not, to my knowledge do that with the Lombardi paper - she simply looked for the gag and env genes from prostate cancer samples - and they there were in spades. I think the search for more genetic variability bypasses the fact it wasn't necessary in the original paper.

We'll see where the science takes us.

This is really difficult stuff and if this is how it all turns out - no one is to blame. These are obviously incredibly difficult subjects which good labs have gotten wrong before - and Silverman's and Dr Ruscetti's and by implication the Dr. Mikovit's are obviously some of the best.

I imagine that, however, XMRV turns out - the researchers will be pointing to it and thinking about it for a long time. The possibility that it was created in a lab has got to really send some minds turning.
 

acer2000

Senior Member
Messages
818
Both her blood and tissue results are consistent with Emory's macaque findings
where XMRV disappeared from blood rather quickly and was later found in organs...

Yep... and still nobody has proposed a study on tissues in humans with CFS. I know blood is easier to test, and I know some people have found it there, but come on... at this point its pretty obvious based on the monkey study someone needs to do tissues.

The CDC study with prostate cancer also is consistent. Switzer also found it in prostate, but not in the blood.

That and the following need to be addressed:

- Differences in positive rates between patients and controls in the non 0/0 studies. If contamination is responsible, how can the results line up so neatly in favor of patient positives when both sets of samples were run through the same reagents/cell lines/etc.. There may be a good reason for this, but it needs to be figured out.

- Nobody has "exactly" replicated the methods from the original paper. It doesn't matter that other labs think they might have a different/better process. At this point, someone needs to go back to square one and do an exact replication. Any other method just adds to the confusion. Details matter. We have to crawl before we walk. And we have to walk before we run.

- Some patients who have taken ARVs (such as Dr. Jones, and Dr. Snyderman) are clearly doing better and (at least in the case with Dr. Snyderman) his immune parameters and cancer markers are improving. This needs to be explained. If not XMRV, what are these ARVs treating? Do they have NK cell counts as well as immune markers from various points on treatment?

- The recent paper from the WPI regarding the immune parameters that correlates 95% with the XMRV positivity. I'd like to see them go further and correlate it with NK cell function.

- The antibody tests need further work. Why are some people PCR/culture positive but antibody negative? Why is the opposite sometimes true? If not XMRV they are picking up, what are they picking up?

Also - we know from the monkey study that immune stimulation draws XMRV back out into the blood. We also know from experience that exercise exacerbates the symptoms of ME/CFS. Why not do a study where you test from XMRV (using the proven methods) following either of these events? Would that not increase the theoretical likelihood of finding it?

Well.. that's what I'd do. :)
 

eric_s

Senior Member
Messages
1,925
Location
Switzerland/Spain (Valencia)
I think my statement was misinterpretated by someone to suggest that I believe this finding rescues XMRV from the plight it is in with CFS. I don't. I think XMRV's connection to CFS rests in labs finding it in the blood of people with CFS - not in their tissues. My personal opinion is that - after a year and a half of searching for it - that that's not going to happen. The most likely scenario IMHO is that Silverman will announce at the conference that the 22RV1 cell line contaminated his prostate samples thus probably providing a smoking gun pointing at the WPI.

On the other hand Silverman could say that the opposite; that his prostate findings are real and that 22RV1 did not contaminate his samples. Since there is new evidence pointing to XMRV being an genetically distinct infectious virus - I think its possible that Dr. Mikovits picked up some of that if she is using tests that search for an array of viruses. She did not, to my knowledge do that with the Lombardi paper - she simply looked for the gag and env genes from prostate cancer samples - and they there were in spades. I think the search for more genetic variability bypasses the fact it wasn't necessary in the original paper.

We'll see where the science takes us.

This is really difficult stuff and if this is how it all turns out - no one is to blame. These are obviously incredibly difficult subjects which good labs have gotten wrong before - and Silverman's and Dr Ruscetti's and by implication the Dr. Mikovit's are obviously some of the best.

I imagine that, however, XMRV turns out - the researchers will be pointing to it and thinking about it for a long time. The possibility that it was created in a lab has got to really send some minds turning.

I can't agree. If even some groups that have found XMRV and say it's a real virus that's in the population (like Switzer's and Singh's) say it's not in the blood or they can't find it there, then i don't think one should use a different standard for ME/CFS. Why look in a way that seems to be the least good? Ok, it is true that the positive ME/CFS studies have reported finding it in the blood and if those were wrong then why should it be in ME/CFS subjects at all? Maybe because it's a type of virus that would fit. And even if there were no reasons i would look.

I find it rather unlikely that Silverman will distance himself from his prostate cancer findings, but we will see. Maybe you have more information than me. What i don't see is how this would be a smoking gun in relation to the WPI's findings. Haven't they said that there never was any 22RV1 in the state of Nevada?

What do you mean with "genetically distinct" virus?

Unfortunately, it does not seem to be only science that is influencing the course of things, in my opinion, so we have to be careful and keep our eyes open. And even more importantly, develop better capabilities to influence things ourselves.

As far as who would be to blame if it was to turn out that the WPI was wrong, i personally would not let them get away as easily. But this is not what has happened and not what i expect to happen, so that's strictly hypothetical. In fact it's the 20th and so i will even donate to them today ;).

I agree that XMRV will not be something that will be forgotten quickly, no matter how things go on from here.
 

jstefl

Senior Member
Messages
250
Location
Brookfield, Wisconsin
This whole XMRV in the blood confuses me greatly.

There seems to be at least some agreement that XMRV exists and can be found in the prostate or other tissues. My question is how did it get there if not thru the blood?

I don't see how the virus could enter directly into the prostate from outside the body, so is there any other path it could have taken?

It seems to me that Dr. Singh, and many others need to answer this question.

John
 

Gemini

Senior Member
Messages
1,176
Location
East Coast USA
and still nobody has proposed a study on tissues in humans with CFS...The CDC study with prostate cancer also is consistent. Switzer also found it in prostate, but not in the blood.

Excellent point. Has anyone requested, in writing, CDC do ME/CFS tissue work? If so, perhaps they could post the CDC's response? Be interesting to see what their position is.
 

LJS

Luke
Messages
213
Location
East Coast, USA
Yep... and still nobody has proposed a study on tissues in humans with CFS. I know blood is easier to test, and I know some people have found it there, but come on... at this point its pretty obvious based on the monkey study someone needs to do tissues.

I do not think this is a valid argument, the original science paper found it in blood and the WPI continues to say they can find it in blood. Heck look how many people come back positive through VIPdx with a blood test, we are not sending tissues off to them. This is getting a little ridicules at this point. If others can not find XMRV in blood it does not mean that it is hiding in tissue; it means the WPI is wrong and their lab is contaminated. If you look at the sequences the WPI posted some are 99.9% identical to 22Rv1 and 99.4% identical to VP62, the WPI are not finding something radically different then what all the tests are designed to find. It would be easy to argue from the sequences the WPI published that they are in fact picking up a lab contaminate due to the fact they are almost identical to the lab strains of XMRV. The Singh lab should have been able to easily find XMRV, if it was there, given the WPI sequences.
 

eric_s

Senior Member
Messages
1,925
Location
Switzerland/Spain (Valencia)
No, it just means that after a certain point others can't find it in blood. Maybe contamination is the more likely explanation (i don't share this view though), but it might also be that the others just are not good enough at looking.

And don't forget some others have found it too. Lo, Hansen and IrsiCaixa. Also RedLabs is finding it and even though they have a license, they are an independent lab from VIP Dx. Hopefully now Dr. Bieger has also found the virus, we should hear about the results any minute now, i guess.

Probably there's much more of it somewhere in tissues and so this might help the others finding it, as it's easier there.
 
Messages
13,774
I guess it's possible that the WPI's science paper on finding it consistently in the blood was wrong, and the result of contamination... but that XMRV is coincidentally related to CFS and needs to be looked for in tissue samples. The would be funny, but is a bit of a long shot.

With that exception, I agree that it's the ability to find the virus in the blood of CFS patients that made us think there's an association, and without that, there's no reason to think that there is an association. It would be good to hear news from the BWG soon. Their last set of results didn't really do much to clear matters up for us.