Due to the following fact - is contamination even possible?

[omerbasket]

Alex, I don't know about the CDC test, but the WPI's and FDA's test did use an annealing temperature (and probably other things as well) that allowed them to find viruses that are not identical or almost identical to XMRV VP62. Had the FDA used a method that focused on specificity instead of sensitivity, they would probably find nothing, and then it's possible that you would have heared Dr. Lo and Dr. Alter supporting the contamination theory. See, it's those very small things that makes this huge difference.
The WPI sequenced every PCR PRODUCT that they had (meaning - everytime that the PCR came up positive - they sequenced the PCR product, which is just a part, pretty small part, of the virus), and also sequenced full XMRV genomes from 2 of the positive patients, and one partial genome (which I guess was much bigger than a PCR product) of a third patient. In the full genomes, they found that they are different from VP62 in just 6 nucleotides - out of 8,185 nucleotides - meaning that it is more than 99.9% identica to VP62, although not 100% identical.
However, these are only two full genomes out of 107 people in which they found evidence for XMRV/MRV infection (not including the britain study and others). So, at first it seemed that they found things that a test that would be very specific to VP62 would find - but now it doesn't seem that way, and although it was probably not expected, it still is reasonable, as they had then only 2 full length sequences.

However, I have to explain - as far as I understand it, by sequencing their PCR products they are making sure that the things that they find are XMRV or at least MRVs, and not, for example, EBV... So there is absolutely no need to be worried about that - that is what is done when you do nested PCR.

 

[kurt]

Hi everyone,

There are two words that I don't see enough in this argument: specificity and sensitivity.

Alteration of PCR parameters, even when using the same test, can affect both sensitivity and specificity. Typically as sensitivity goes up ("did we find anything") specificity goes down ("did we find the one thing we were looking for"). Alteration of such things as reagent concentrations and PCR annealing temperature are just two factors I am aware of that affect this.

Here is one solution to the problem, and in this nearly ALL the data is correct, potentially:

The CDC could (not do, has to be proved) have low sensitivity and high specificity. They are using a very specific test looking for a known strain of XMRV. It isn't there.

Lo and Alter, plus WPI, have learned (I suspect, not proven) to adjust parameters for sensitivity not specificity. Indeed, now they use parameters that will detect PMLVs and not just XMRV. So they find what is there, but it might not be anything like the original XMRV strain - indeed they may be finding multiple strains and related but different viruses, as they have claimed.

The CDC is operating from the assumption that there is a very narrow range of genetic diversity in the target virus. The WPI are operating from the assumption that there is a huge genetic diversity in the target virus family. These assumptions drive different parameter selections.

So the CDC can't find anything, and the WPI is finding it all over the place, even with the "same" test. This is just an hypothesis, but it explains the data. All the labs need to actually sequence the viral strain they are finding - just saying it is there is not enough. I do realize this uses up resources, and there is not enough funding - so the blame for this is still lack of funding in my view.

Coffin's hypothesis of a culture origin of XMRV is something else - personally I suspect it was infected by an XMRV positive lab worker, and did not originate there, but I do think it likely that he found two murine endogenous viral strains that may be related to the viruses that did give rise to XMRV, originally.

Bye
Alex

I think that is an interesting theory. However, the CDC actually used the WPI test and found nothing. So this can't be just a specificity/sensitivity issue. All the labs that have run PCR tests for XMRV have been competent enough to calibrate their tests successfully, which means they know how to adjust sensitivity and specificity. Also this means they CAN find the strains of XMRV that WPI found and described in the Science article.

There is some explanation for the diverse findings, but I am not convinced anyone has actually figured out yet what is happening. In prior retroviral hunts there have been cases where control samples tested right (negative) and patient samples tested wrong (got false positives). There are various testing artifacts that can influence this type of testing.

My own theory (unproven but I think credible) is that some trait in CFS blood is interacting with the WPI's tests for some reason besides XMRV. We already know that CFS patients have several abnormalities in their blood, and there might even be a common CFS co-infection with genetic sequences that is interacting in some way with some of the tests but not others, due to test design. After all we do have loads of co-infections, and a messed up immune system. No doubt we have a variety of undiscovered sequences in our blood.

There is no question contamination has been involved in some of the testing, as labs have changed reagents and stopped the contamination. And there is no question that highly specific and sensitive tests fail to find the XMRV sequences in CFS samples in the majority of studies to date. The fact that diverse results are perplexing does not prove anything about XMRV in CFS, except that we do not have all the answers yet.

 

[alex3619]

Had the FDA used a method that focused on specificity instead of sensitivity, they would probably find nothing, and then it's possible that you would have heared Dr. Lo and Dr. Alter supporting the contamination theory.

Hi omerbasket, thank you for your reply. Indeed the FDA/NIH did look at a less specific but more sensitive test, for which we can be thankful. However, they could not find XMRV until they increased the sensitivity even further - and I think this is still unpublished.

I was already under the impression that the entire virus was not sequenced in most cases - possibly because of the issue with small sequences being amplified in nested PCR, the real virus is still scarce and only this specific fragment is amplified and abundant enough for sequencing. I have not studied the genetics of specific primers much to determine if they may have problems, maybe someone else can comment. Generally it is considered reliable for well understood viruses.

My understanding is the CDC test was optimized for the reference clone - it could be highly specific, and only sensitive to that one strain. Whether it is or not remains unknown though.

We need the full *MLV genomes from every patient we can afford to test - for one thing I am fairly confident the genetic diversity will be much higher than so far established, presuming that XMRV really is associated with ME/CFS. Indeed, I think that some of this testing may be in the as yet unpublished WPI results. The lack of published genetic diversity is part of what is hurting XMRV credibility - small amplified viral segments are not enough. Only two patients with virus fully sequenced, is not enough. If many viral strains are sequencing the same in large numbers of patients, then there is a problem with the WPI results. If they are very different (and my understanding is that the unpublished data shows this) then the contamination theories are in big trouble. I do understand that finding enough of the virus to fully sequence is a major problem though - the resources including money this requires is huge, so yet again I blame funding.

This of course leads to the wider debate about XMRV/MLVs - it is still possible that XMRV is rare and not very relevant, but that the PMLVs are the real issue - for which contamination arguments have still not got any credibility. The main risk for contamination is murine cells, but they routinely test for this, sometimes using different tests just to be sure.

To me it would also make sense to sequence the viral proteins - does anyone know if this has been done extensively enough so we can get some idea of the diversity from the proteins? The quantity of viral proteins that has been isolated should be enough for amino sequencing. There is some risk though that the proteins may be more highly conserved than the genetic sequences.

I also also wonder if enough anti-XMRV goat antibody can be made, why this hasn't been used to extract live virus from patient samples? Is it funding?

So, the main point I would like to recap - the contamination theories do not hold against a diversity of MLVs. The argument this is based on, which I think Coffin refered to at SOK, is that such contamination is routinely found. However, that is the point - the testing regimes find it. This has not been found in routine testing by WPI, Lo or Alter.

Does anyone know what is happening with the Singh research?

Bye
Alex

 

[alex3619]

I think that is an interesting theory. However, the CDC actually used the WPI test and found nothing. So this can't be just a specificity/sensitivity issue. All the labs that have run PCR tests for XMRV have been competent enough to calibrate their tests successfully, which means they know how to adjust sensitivity and specificity. Also this means they CAN find the strains of XMRV that WPI found and described in the Science article.

Hi Kurt, you are making my point for me. It is presumed the CDC used the same test. Where is it stated (commentary, publication, public announcement) that for example magnesium concentration and annealing temperatures were identical? They were optimized to a reference strain - such optimization suffers from the Hill Climbing problem well know in optimization theory - they gravitate to local maxima/minima. So optimized yes, but optimized to what? How you measure the optimization and what you account for will determine the final specificity and sensitivity. The tests will not be identical if separately optimized. Finding a reference strain only proves you can find a reference strain.

The notion that such optimization is ideal and works universally was abandoned in computer science in the sixties if I recall correctly. It is simply wrong. The best you can hope for is "kind of" optimized.

Successful over-optimization to a reference strain could reduce the sensitivity to variants to almost nil. This is also well known in computer science. It will however give their test a good level of specificity, with perhaps a high sensitivity to that specific strain.

Indeed in my neural network research it was important to avoid highly specific optimization, as this resulted in a reduced capacity to generalize the result. Please note I am not saying that the CDC does have this problem, only that it could. It is not proven, only an hypothesis. So every new test has a chance of being optimized correctly - some will, some wont, so some will find XMRV and some wont. Which is what we see.

All of the issues I raised could be cleared up by careful testing, calibrating their tests to multiple pathogen strains, and probably other methods etc. Nobody has done this (except of course WPI, Lo, Alter).

Bye
Alex

 

[alex3619]

My own theory (unproven but I think credible) is that some trait in CFS blood is interacting with the WPI's tests for some reason besides XMRV. We already know that CFS patients have several abnormalities in their blood, and there might even be a common CFS co-infection with genetic sequences that is interacting in some way with some of the tests but not others, due to test design. After all we do have loads of co-infections, and a messed up immune system. No doubt we have a variety of undiscovered sequences in our blood.

Hi Kurt, this is indeed a credible hypothesis. For example, the ciguatera antibodies were finding cardiolipin or something, not ciguatera in the ME/CFS patients. Antibodies look for similar amino acid structure - they don't care what the substance is.

One of the reasons I find your argument credible is that we don't know how much transient and short lived DNA/RNA is entering the blood from destroyed gut bacteria. It should not happen, ever, but our gut integrity is suspect. While cross-contamination from our own DNA is unlikely in PCR, there might be specific sequences from gut bacteria. Here I also include gut bacteriophage nucleic acids - in other words viral sequences.

So your hypothesis has some merit.

Bye
Alex

 

[Bob]

My own theory (unproven but I think credible) is that some trait in CFS blood is interacting with the WPI's tests for some reason besides XMRV. We already know that CFS patients have several abnormalities in their blood, and there might even be a common CFS co-infection with genetic sequences that is interacting in some way with some of the tests but not others, due to test design. After all we do have loads of co-infections, and a messed up immune system. No doubt we have a variety of undiscovered sequences in our blood.

I've been wondering if this might be a possibility as well... Because of the low specificity of the WPI's test, I was thinking that they might be detecting sequences from viruses other than XMRV, such as other MLV-related viruses... There might even be a number of different MLV-related viruses involved... It might explain why some researchers can't find XMRV and the fact that the WPI have sequenced whole XMRV genomes, if XMRV is just one of a number of MLV-related viruses involved in CFS/ME.

 

[omerbasket]

the CDC actually used the WPI test and found nothing.

Where is it stated? Can you also quote the sentences from where it is stated?

 

[omerbasket]

I've been wondering if this might be a possibility as well... Because of the low specificity of the WPI's test, I was thinking that they might be detecting sequences from viruses other than XMRV, such as other MLV-related viruses... There might even be a number of different MLV-related viruses involved... It might explain why some researchers can't find XMRV and the fact that the WPI have sequenced whole XMRV genomes, if XMRV is just one of a number of MLV-related viruses involved in CFS/ME.

Bob. I think you are correct - but Kurt, I think you aren't. The method that the WPI uses can, to my understanding, find MLV-related viruses and think that those are XMRVs - because in the first study they found 68 of their patients to be positive for a not-very-much-specifc test for XMRV, and when they sequenced two of them it was XMRV - so that guessed that a test designed for XMRV (but that can also find things that are just close to XMRV, but aren't XMRV VP62) and that brings 2 out of 2 full genomes that are XMRV, found XMRV every time it came up positive. It might not be so, I guess - but their test IS designed to only find XMRV or realted sequences, and they also sequence every PCR product, so they should only find MRVs, and not something that is entirely different (like HHV-6, for example). Add to that their antibody test, that showed that 50% of the patients tested, which were positive by PCR, had antibodies against a gammaretrovirus (as opposed to none of the controls) and you can understand that they are finding MRVs anyway - and whether it's XMRV or other MRVs, perhaps they don't know - but currently, it's much more important to know if the patient carries an MRV, any MRV, than if it's XMRV or another MRV - and therefore the sensitivity is more important than specificity, as long as you make sure that in the end, if what you found wasn't a MRV, you would know that and not consider it positive - and the WPI are making sure of that. That is why they are sequencing every PCR product, which is very important (and they know that...).

 

[Snow Leopard]

My own theory (unproven but I think credible) is that some trait in CFS blood is interacting with the WPI's tests for some reason besides XMRV. We already know that CFS patients have several abnormalities in their blood, and there might even be a common CFS co-infection with genetic sequences that is interacting in some way with some of the tests but not others, due to test design.

That potentially explains the confusing serology results. But not the fact that even the scientists in the contamination camp do not agree on what exactly is the source of the contamination. The Retrovirology studies for example did not agree with one another.

I still am increasingly leaning towards 'contamination', but there is more science to go before 'contamination' is proven.

 

[omerbasket]

Again, the serology done by the WPI would have found only a gammaretrovirus, to my understanding, and nothing that is not a gammatertovirus. And if it's that gammaretrovirus or another one, is much less important. There are strains of HIV-1 which are "only" 84% identical to one another, there are strains of HCV which are "only" 79% identical to one another. For comparison, the sequences found by the NIH/FDA, which are refered to as PMRVs, as opposed to XMRV, are 84%-96% identical to XMRV VP62. As Dr. Mikovits said at the state of knowledge meeting, it's the first time in virus history that we take a single strain of virus and say that everything which is not at least 99% identical to it is not the same virus. Again, you can look at HIV-1 and HCV as examples as to how right she is.

Besides that, also their PCR assay wouldn't detect things that are not MRVs. I mean, it would, at first - but they sequence every PCR product that they get, and then they know whether it's a MRV or not (if it's not, then they DON'T consider it positive, ofcourse). And besides that, they have published two full genome sequences of viruses isolated from ME/CFSpatients, that are more than 99.9% to XMRV VP62.

 

[Mark]

Back when the Science paper came out, and ever since then, I too have pondered the type of hypothesis that Kurt sketched, whereby something in CFS patients' blood is responsible for the results, and I still think that something along those lines is quite plausible as an explanation of the conundrum. A related hypothesis: after watching the SoK workshop, one thing that jumped out at me was multiple researchers reporting surprising findings of fragmentary proteins and sequences presumed to be 'bits of dead cells' floating around in the blood - so something along those lines might also be a confounding factor: such fragments may conceivably be able to recombine with other fragments during PCR.

These sort of hypotheses relate to the suggestion I made recently elsewhere about Dr Alter's King Solomon reference - he may have been suggesting that if the world is unprepared to call whatever the WPI found "XMRV", perhaps their findings might be rescued by abandoning that specific theory...though I don't personally think so, because I don't see the rest of the world champing at the bit to investigate whether there's any other evidence for slightly different HGRVs in ME/CFS patients...

Hi Kurt, you are making my point for me. It is presumed the CDC used the same test. Where is it stated (commentary, publication, public announcement) that for example magnesium concentration and annealing temperatures were identical? They were optimized to a reference strain - such optimization suffers from the Hill Climbing problem well know in optimization theory - they gravitate to local maxima/minima. So optimized yes, but optimized to what? How you measure the optimization and what you account for will determine the final specificity and sensitivity. The tests will not be identical if separately optimized. Finding a reference strain only proves you can find a reference strain.

What this point reminds me of, is the CDC's recent comments (at CROI?) regarding the possible infection of some of their lab workers. If I have this right: somebody from the CDC (Schwitzer?) commented that in the early stages of developing their XMRV test, they had found a few 'positives' in their controls - for which they used their own lab workers - but had assumed these positives to be false positives (since they were found in controls) and adjusted the sensitivity of their tests accordingly.

The whole thing sounded extraordinary to me at the time, and still does: "Oh, by the way, actually we did find a whole load of XMRV+ on our early tests, but assumed they were errors and recalibrated until they were negatives...and we didn't think this was worth mentioning until now...". Irony of ironies if XMRV infection has spread via the lab after being created in the lab, has infected many CDC lab techs, and then scuppered the CDC's test because they used those infected lab techs as controls...

 

[asleep]

I think that is an interesting theory. However, the CDC actually used the WPI test and found nothing. So this can't be just a specificity/sensitivity issue. All the labs that have run PCR tests for XMRV have been competent enough to calibrate their tests successfully, which means they know how to adjust sensitivity and specificity. Also this means they CAN find the strains of XMRV that WPI found and described in the Science article.

Kurt, you are making claims here without any evidence to support them. Sure, the CDC used the WPI primers, but there is no evidence that they achieved the sensitivity of the WPI. Because their positive control was a VP62 spiked sample, the only claim they can make is that their assay can detect VP62. Namely, they have only demonstrated specificity.

All of the negative studies have conspicuously avoided demonstrating their sensitivity by avoiding the use of positive clinical samples. Therefore, none of them can make any honest claims about sensitivity.

Now, I know you will fire off the stale rallying cry "But the positive clinical samples are unvalidated, and we can't be sure they are actually positive." This is an unconvincing and diversionary argument for a number of reasons:

Firstly, it is fundamentally unscientific by requiring proof as a prerequisite for attempting investigation. Something that is "unvalidated" can still lead to meaningful results. For example, if an assay calibrated to "unvalidated" positive clinical samples can consistently show substantially higher positive rates in patient vs control samples (under controlled conditions), then it doesn't matter if the calibration samples were "validated." One can argue about what exactly the assay is picking up, but there can be no argument that something is there and that the calibration samples are likely positive for this something. Avoiding this approach entirely screams "We are not interested in finding anything."

Secondly, it is circular reasoning that, in effect, leads to strict avoidance of actual replication. A variation of this argument could essentially be lobbed at any new discovery as a convenient excuse for avoiding replication. E.g. I could report that I found a monster turtle in a specific pond. Someone could then say, "That report is unvalidated and we don't know if monster turtles even exist!", then wonder off to a completely different but similar pond, find nothing, and say "See, if monster turtles really existed, they should have been found in this other pond." It should go without saying that something has been missed here!

I have bolded two examples from your post (above segment and last segment below) where you casually assert that the negative studies demonstrated appropriate specificity and sensitivity. Again, without evidence of this, you are arguing from assertion and making very misleading statements.

There is some explanation for the diverse findings, but I am not convinced anyone has actually figured out yet what is happening. In prior retroviral hunts there have been cases where control samples tested right (negative) and patient samples tested wrong (got false positives). There are various testing artifacts that can influence this type of testing.

Proposing an unsubstantiated possibility does not constitute a counter-proof. There is no direct evidence that the WPI or Lo/Alter have false positives. If you want to claim that their results are false positives, the burden of proof is on you.

My own theory (unproven but I think credible) is that some trait in CFS blood is interacting with the WPI's tests for some reason besides XMRV. We already know that CFS patients have several abnormalities in their blood, and there might even be a common CFS co-infection with genetic sequences that is interacting in some way with some of the tests but not others, due to test design. After all we do have loads of co-infections, and a messed up immune system. No doubt we have a variety of undiscovered sequences in our blood.

Again, proposing an unsupported possibility as a counter-argument. The WPI's results could also be due to invisible pixies sprinkling XMRV-like angel sparkles predominantly in the patient samples.

As with your previous paragraph, this is actually a textbook example of pseudoskepticism. I suggest that people read this article in order to understand some of the common, fallacious tactics on display by those arguing against XMRV. In Kurt's post, in particular, we find:

• Assuming criticism requires no burden of proof: e.g proposing that the WPI and Lo/Alter results are "false positives" without any direct evidence.
• Making unsubstantiated counter-claims: e.g. asserting that the negative studies have demonstrated sensitivity without any evidence.
• Counter-claims based on plausibility rather than empirical evidence: e.g. claiming that the positive results are due to some potentially plausible theory without any direct evidence that said theory is actually producing the observed results.

There is no question contamination has been involved in some of the testing, as labs have changed reagents and stopped the contamination. And there is no question that highly specific and sensitive tests fail to find the XMRV sequences in CFS samples in the majority of studies to date. The fact that diverse results are perplexing does not prove anything about XMRV in CFS, except that we do not have all the answers yet.

You are implying an unjustifiable extrapolation of isolated incidents (obvious cases of contamination by Huber, etc) to other cases, again without evidence. These obvious contamination cases don't even outwardly look like the WPI and Lo/Alter studies (in that they showed positive results equally in controls and patients or in more controls, unlike the positive studies that showed consistently and starkly larger positive rates in patients than controls). Again, possibility (sans evidence) does not equal evidence or proof.

Lastly, your appeal to "perplexing" results (based in large part on selective analysis and fallacious arguments, as I've pointed out here) strikes me as an attempt to incite artificial confusion and doubt.

 

[Bob]

Can anyone help me with this please?...
When people say that the WPI are "sequencing every PCR product"... Does that mean that the WPI are sequencing every protein/DNA/RNA sequence that they detect with PCR?

 

[omerbasket]

As far as I understand it:
When they do PCR and come up with a positive result, the positive result is because the PCR found a chain of nucleotides. This chain, I think (but don't know), is about a couple of hundreds of nucleotides long. Then, they sequence that - so they get, for example, a strain of 300 nucleotides. Than, they compare it to the related chain in the virus they are looking for, and see if it fits, or nearly fits. If so - than it is a true positive (from contamination or not, that's not the issue here... They check for contamination via other routes), as opposed to something which is entirely different, like a human DNA chain. So, that is how they see that they are looking at XMRV or other MRVs (or MLVs) as opposed to, for example, CMV.

 

[insearchof]

Stand out Monkey

A short diversion, someone earlier in the thread mentioned the Rhesus Macaques study. Is there a thread here devoted to a discussion on that paper, because I cant seem to find one. I seem to recall having read details on it (possibly from the transcript of the FDA BWG) earlier and recall that there was one monkey in which they found no viremia in its sera, even though it was infected with XMRV. I am sure though that this same monkey produced an antibody response and that they found XMRV in the tissues.

So I am wondering, did this monkey hold answers as to why XMRV is not always detectable in sera? Did they take a closer look at this monkey? What could have been going on in the sera of that one monkey? Have they considered doing a larger study with Rhesus Mcaques to explore this? Does anyone know?

 

[acer2000]

A short diversion, someone earlier in the thread mentioned the Rhesus Macaques study. Is there a thread here devoted to a discussion on that paper, because I cant seem to find one. I seem to recall having read details on it (possibly from the transcript of the FDA BWG) earlier and recall that there was one monkey in which they found no viremia in its sera, even though it was infected with XMRV. I am sure though that this same monkey produced an antibody response and that they found XMRV in the tissues.

So I am wondering, did this monkey hold answers as to why XMRV is not always detectable in sera? Did they take a closer look at this monkey? What could have been going on in the sera of that one monkey? Have they considered doing a larger study with Rhesus Mcaques to explore this? Does anyone know?

In short yes... they infected the monkeys with XMRV and it only transiently existed in the blood. It persisted in the organs such as the liver, spleen, lungs, prostate, GI tracts, etc... and could be provoked back into the blood by triggering an inflammatory/immune response. Which frankly, at least on the surface, sounds pretty much like how most patients experience the illness. A mouse model recently showed very similar findings. So this could be a reason why they aren't consistently finding it in the blood and a good reason to move onto a study that looks in those tissues. Why they haven't done this is beyond me... Doctors seem to have this inappropriate belief that if they can't find something in the blood, then its not there... which of course is completely silly to say the least.

Another suggestion would be of them to test patients after exercise. It seems logical that if CFS patients are made worse by exercise and then experience PEM, perhaps testing them for XMRV (and other things) during this PEM period would be logical. I haven't heard of anyone doing that though... again - seems obvious.

 

[insearchof]

Hi Acer2000

Acer, I am aware of the transient existence in the sera generally of the monkeys, but I am sure I read that there was one monkey where they found nothing in the sera - nothing at all. Not that it was there, but only for a short period, like witnessed in the other monkeys. There was nothing found!

It seems very strange that they could not find anything in the sera of this one monkey, which is why I made the post.

Perhaps I misread the finding, but I dont think so - but as I read it a while a go, I cannot discount that possibility, which is why I wanted to read the paper. So if anyone has access to it and can provide a copy, I would be very grateful.

 

[lansbergen]

Hi Acer2000

Acer, I am aware of the transient existence in the sera generally of the monkeys, but I am sure I read that there was one monkey where they found nothing in the sera - nothing at all. Not that it was there, but only for a short period, like witnessed in the other monkeys. There was nothing found!

It seems very strange that they could not find anything in the sera of this one monkey, which is why I made the post.

Perhaps I misread the finding, but I dont think so - but as I read it a while a go, I cannot discount that possibility, which is why I wanted to read the paper. So if anyone has access to it and can provide a copy, I would be very grateful.

Do you mean this one? Infection, viral dissemination and antibody responses of Rhesus macaques exposed to the human gammaretrovirus XMRV

Next an initial set of 3 monkeys were inoculated with XMRV VP62, a molecular clone isolated from the prostate of a human patient (4) and followed for plasma viremia using quantitative RT-PCR (Figure 2a). Among the 3 animals, plasma viremia was detected in 2: one male (RIl-10) starting on day 4 post infection (pi), with a peak at 7500 viral copies on day 7, decreasing to below the detection levels by day 14 (Fig. 2b) and a female (RYh-10), who showed a delayed and lower acute viral replication kinetic at days 14-20. The third animal (a male, RLq-10) never showed vRNA levels above the detection threshold. However, proviral DNA was detectable in all 3 animals during the initial 3-4 weeks pi in blood (Fig. 2c), confirming the susceptibility of macaques to XMRV infection.

 

[Bob]

As far as I understand it:
When they do PCR and come up with a positive result, the positive result is because the PCR found a chain of nucleotides. This chain, I think (but don't know), is about a couple of hundreds of nucleotides long. Then, they sequence that - so they get, for example, a strain of 300 nucleotides. Than, they compare it to the related chain in the virus they are looking for, and see if it fits, or nearly fits. If so - than it is a true positive (from contamination or not, that's not the issue here... They check for contamination via other routes), as opposed to something which is entirely different, like a human DNA chain. So, that is how they see that they are looking at XMRV or other MRVs (or MLVs) as opposed to, for example, CMV.

Thanks omerbasket.
I didn't know that the WPI have sequenced everything that they have detected in PCR tests.
I didn't know that was how the WPI were carrying out their work.
It is reassuring to know, seeing as their PCR testing is set to be more sensitive and less specific.
What Judy was saying at The State of the Knowledge conference, about detecting other viruses with her less-specific PCR test, makes sense to me now: Her PCR detection of XMRV is valid (even though her PCR detects other viruses) as she has sequenced each 'product' that she has detected with PCR in order to confirm that it is indeed XMRV.
The WPI's more-sensitive PCR methodology seems to be an obvious possible reason why they may be able to detect XMRV, unlike most other researchers. I believe that Lo followed Judy's methodology very closely and then he detected the PMRV's.

 

[omerbasket]

You should also be aware that every single time that the WPI finds a positive for XMRV via PCR (and they probably found about a 1,000 until now) not only sequence the PCR product, but they also isolate an infectious virus from the sample.
As Dr. Mikovits said at the NIH conference, they also check their cell lines (in which they culture the virus) for mouse DNA contamination every week - and the tests always came back negative. Moreover, when their PCR test comes up positive - they confirm it with at least one other method to be sure that it's a real infection.

They also checked every positive sample from the "Science" papaer for mouse mtDNA (using William Switzer's test) and it always came back negative.