XMRV not found in Fibromyalgia Patients in Spain

[Esther12]

It is very easy to rule out contamination: ensure that all case and control specimens are collected in the same manner and fully blinded before any processing and testing. If, under such conditions, the same positivity rates hold (~4% controls vs ~80%+ cases), then contamination is probabilistically ruled out.

Have we actually had that yet? I think the WPI's UK study collected patient and control samples differently, as did the Alter paper. Maybe this sort of exact blinding isn't as normal as we'd assume?

 

[asleep]

I think the WPI's UK study collected patient and control samples differently, as did the Alter paper.

Is this definitely true? Even if it is, the Alter paper has already been published and the WPI's UK study is just one example from well before the whole contamination hoopla.

I've heard (though cannot verify) that Cheney did a very simple validation of the Science paper by sending blinded samples to VIPDx. Assuming consistent collection methods, surely this sufficiently controls for contamination. Yet, supposedly no one will publish...

 

[Ernie]

Well my diagnosis is fibromyalgia and I am seropositive and culture positive.

 

[Esther12]

Is this definitely true? Even if it is, the Alter paper has already been published and the WPI's UK study is just one example from well before the whole contamination hoopla.

I've heard (though cannot verify) that Cheney did a very simple validation of the Science paper by sending blinded samples to VIPDx. Assuming consistent collection methods, surely this sufficiently controls for contamination. Yet, supposedly no one will publish...

The UK study's not been publshed, so I'm not sure where to look to back this up, but at the time it was presented at the XMRV conference it was reported here that the samples were collected differently (to focus on the illest patients the WPI arranged for home collection etc...).

The sort of simple testing Cheney did does sound like a sensible thing to do (and should have been the first step 18 months ago imo!).

 

[asleep]

PCR is complicated

I will remind people of this study that has been out awhile now, which indicates that XMRV is very difficult to find with PCR:

Detection of Xenotropic Murine Leukemia Virus-Related Virus in Normal and Tumor Tissue of Patients from the Southern United States with Prostate Cancer Is Dependent on Specific Polymerase Chain Reaction Conditions

To quote from the study:

Additionally, the failure to detect XMRV may be attributable to differences in the detection techniques employed. We have found that detection of XMRV required rather specific conditions.

Thus, there is much more to PCR than just primer selection.

 

[Cort]

And even if some or most groups did, my understanding of PCR (as a non-scientist) is that there are many more factors involved than just primers. PCR is a complex chemical reaction and any number of variables can throw it off. Therefore, any PCR attempt must be empirically validated against know clinical positives, which is a step that none of the negative studies have done. And that is to say nothing of potential cohort, collection, storage, processing, tissue reservoir, replication cycle, etc issues.

I have been told that labs are now growing XMRV and using the live virus to produce their tests - so that will e really helpful. I have also been told that labs are starting to replicate the WPI's methods down to a T, to the reagants, test tubes, etc. Cohort selection, storage, replication cycle - all of these could explain reduced levels of positives but none, to my mind, can explain the zero percentages found. If everyone was finding 20% positives we'd be doing great!

Regarding the WPI finding XMRV using PCR, you will recall that Mikovits was only able to find it in a small percentage of samples from a very tightly defined cohort and after testing them multiple times.

This is not true. THey found them in 67% of the patients and only some of them needed repeated testing. The need for repeat testing apparently wasn't even referred to until the WPI did their rewrite of their methods. If it was a major factor I assume it would have been in the original study. I asked Dr. Mikovits i she found them on the first draw and she said yes. That is another reason for reduced prevalence but not for zero prevalence.

The sum of these negative studies amount to very little beyond a lesson on how difficult it is to find XMRV using PCR. Remember, absence of evidence is not evidence of absence. Furthermore, this scientific truth is not conditional on the number of times you fail to find something: absence of evidence x 100000 is still not evidence of absence.

In a world of limited research dollars it actually means quite a bit. Thankfully we have a bevy of really good labs looking for XMRV. This will be figured out.

Also, the idea that PCR was central to either the findings or the publicity of the Science paper is pure fantasy.

Have to disagree. PCR is how researchers find viruses now...That and the antibody findings are the two key parts of the paper. Notice that every research paper on XMRV reports its PCR findings first. Notice that was what the WPI lead THEIR paper off with...PCR was the key finding for the research community.

But not all approaches are equal, especially if they are fundamentally inadequate or not performed correctly. You seem very fixated on what "generally" works for finding known viruses without any consideration for the possibility that we may be in the midst of discovering the limits of currently favored technology.

That is possible........I keep coming back to the WPI's ability to find XMRV without using any really special methods. It's possible that there's quirk, some factor - some little step you have to do just right - but I've been told if that turns out to be the case it will be an exception. In any case it sounds like true replication studies are being done - which they should be - and hopefully they'll find it.

This is an alarming and profoundly unscientific statement. You cannot bypass replication and declare something dis-proven by accumulating failures of different methods. To suggest otherwise is wholly misleading and logically unsound. The fact that you suggest and condone this idea is shocking. To me, this is nothing short of a public admission that you desire XMRV association to be decided politically instead of scientifically.

So to shock you so badly . I think its just a reality...it has nothing to do with what I desire...if researchers keep poking at it from different angles and none of the angles work eventually they'll decide that they've covered so many options that they just can't imagine XMRV slipping out from under their grasp...In any case - hopefully some true replications are under way..

 

[Angela Kennedy]

True Angela but two points, 1 whats happened to the 3.7% healthy controls infected ? and two Judy Mikovits has suspected many diseases could be xmrv linked, and this was one of them. Didnt know about the diabetes study. again no 4% of healthy infected people ?

Sorry- I was joking about the diabetes study. There hasn't been one.

My problem is how negative studies of other illnesses are impacting on Canadian defined ME/CFS XRMV research.

4% of 'healthy' people being found XMRV positive is not a large amount- which is why the 67% of CC ME/CFS XMRV + is so significant.

What if they did find say, 4% in a cohort of diabetes sufferers, or even 0%? It wouldn't be another 'negative' for CFS, for example. Fibromyalgia, though sometimes found in ME/CFS sufferers (depending on diagnosis though) is not actually CC ME/CFS.

The reason we need to keep this in mind is because, with all the contamination and detection methodology fiasco, it's easy to forget the issue of patient cohort and ME/CFS, and how this important issue can get drowned out.

 

[Cort]

I think this might be the Madrid group that has so far never been able to find XMRV. Nevertheless, for me too, with each negative study it gets harder and harder to think of an explanation why this might be happening.
But then i think that the WPI and Alter/Lo have been working on this for so long now and we haven't heard them say "Oops, sorry, we've made a mistake". So how could it be possible for them to create false positives over and over again at similar rates (i guess) without noticing the mistake? This seems as hard to explain as the discrepancy in results.
I'm looking forward to the webcast by Alter and Lo and other upcoming events like the workshop. But i agree with Esther, how can it be that the question has not been cleared after so long, when there would be a more or less easy way to achieve that? BWG phase III will do that, though.

Whatever happens we will have to fight to get to a solution and i'm convinced we can make it, if we put all we have (i'm not thinking of money exclusively here, not at all) in it. We will have to force it, one way or the other. You can't stop a million of people.

Good points. No one has said they made a mistake. We haven't heard Ruscetti or Silverman or Alter/Lo saying that - which is important. I am of mixed mind on Alter/Lo.. they haven't said no (which is good) but I also really expected them to say 'yes' by now. LeGrice said he thought they would be able to clear up the confusion pretty quickly - but it seems that they, like many of the other groups, are taking longer than expected. On the other hand it may be that this is their first real opportunity to speak about their findings publically....it will be interesting...

 

[eric_s]

I've heard (though cannot verify) that Cheney did a very simple validation of the Science paper by sending blinded samples to VIPDx. Assuming consistent collection methods, surely this sufficiently controls for contamination.

Can you say more about this? It would be very interesting to have such results.

 

[asleep]

Can you say more about this? It would be very interesting to have such results.

I wish I did know more about it, but that is the extent of what I've heard (and as I mentioned, I haven't verified that it's even true). I only brought it up as a counter-example to the idea that contamination fears were the sole force preventing publication of positive studies.

 

[Cort]

Can you say more about this? It would be very interesting to have such results.

If he had patients and controls in there I think that would be significant. And VIP DX's lowered positive percentage vs Cheney's higher positive percentage fits as well; that's what you would think would happen....

It is a muddle for sure..

 

[asleep]

I have been told that labs are now growing XMRV and using the live virus to produce their tests - so that will e really helpful. I have also been told that labs are starting to replicate the WPI's methods down to a T, to the reagants, test tubes, etc. Cohort selection, storage, replication cycle - all of these could explain reduced levels of positives but none, to my mind, can explain the zero percentages found. If everyone was finding 20% positives we'd be doing great!

Actually, I think 0% findings are much easier to explain: their methods couldn't detect wild-type XMRV. Given their use of unvalidated PCR, there are myriad reasons why this could be.

And how are researchers "growing XMRV"? Are they growing it in human cell-lines? If so, wouldn't this, by definition, constitute an infectious human retrovirus (since it is infecting and replicating in human cells)?

This is not true. THey found them in 67% of the patients and only some of them needed repeated testing. The need for repeat testing apparently wasn't even referred to until the WPI did their rewrite of their methods. If it was a major factor I assume it would have been in the original study. I asked Dr. Mikovits i she found them on the first draw and she said yes. That is another reason for reduced prevalence but not for zero prevalence.

I stand corrected on the 67%. But to repeat what I've said elsewhere, the issue is not whether XMRV can be found by PCR (it can), but instead whether it can reliably detect wild-type PCR under unvalidated conditions (it cannot).

In a world of limited research dollars it actually means quite a bit. Thankfully we have a bevy of really good labs looking for XMRV. This will be figured out.

Scientific truth is not amenable to the limitations of research dollars. Any attempt to justify unscientific conclusions on the basis of "limited research dollars" is the stuff of politics and apologetics, not science.

So to shock you so badly . I think its just a reality...it has nothing to do with what I desire...if researchers keep poking at it from different angles and none of the angles work eventually they'll decide that they've covered so many options that they just can't imagine XMRV slipping out from under their grasp...In any case - hopefully some true replications are under way..

You conveniently omit the nagging fact that not one of the "many options" these researchers have covered is a method that has previously been successful in finding XMRV. I'm hard-pressed to imagine an honest researcher who would try everything except that which has worked before and then conclude that nothing works.

 

[MNC]

If I am not wrong this is that second Spanish study that we knew in advance that wouldn't find it.

I can't remember very well now, maybe some other Spaniards can confirm this, but I think that there were two studies announced long ago in Spain:

1- A small one in Barcelona for very few patients (15?) from Can Ruti Hospital (viruses and HIV specialists) with very little money and patients asking for donations in every forum, where Judy Mikovits went personally to supervise and help that we expected that would be positive and indeed it was positive and they found it.

Again, if I am not wrong, our Government has recently denied public funds to them for more research despite these positive findings of XMRV.

2- And the second one in Madrid with public funds with the expected goal of not finding it run by skeptic doctors such as Dr.Gonzalez to whom I met in person and was the biggest &%$%& ever. He for instance denied to take a look at my many medical reports and said it was psychosomatic and prescribed me CBT, relaxation & graded exercise. I still keep his written report saying this.

This is what I remember, but I am very very tired an unwell at the moment too and I could be wrong. Surely other Spanish people could confirm this better than me, but by now I would take these news with a bit of skepticism.

Regards.

 

[free at last]

Qoute
You conveniently omit the nagging fact that not one of the "many options" these researchers have covered is a method that has previously been successful in finding XMRV. I'm hard-pressed to imagine an honest researcher who would try everything except that which has worked before and then conclude that nothing works END

That is true about most negative studys as far as im aware,
but the Kerr study did do a anti body test which concluded that the anti body testing was too cross reactive, although they did detect about 4 samples i think ? that seemed to be specific to xmrv, and got those positive results. Not sure what there explanation for the specific xmrv positive samples was ? but how specific is the WPIs anti body test, does anyone know?

It may be too small a result, but Sing did announce she detected anti bodys to XMRV in one patient sample, when she applied for her patents. Again as far as im aware her test was xmrv specific, If anyone knows if thats so and how reliable it is saying her serology test was specific, can we say that with a 100% certainty ?

Sing seems to be takeing a awfully long time to publish any of her findings. i wonder why ? after some comments i read about a email exchange with one of the researchers, it did not sound encouraging. But since then nothing, not a peep.

Though sings cancer patent statistics did sound very encouraging as far as xmrv being a new human retro virus infecting humans is concernd. Not least of which was the gleason score correlation. something that appears to me to be very unlikely from a contamination, or some other anomaly throwing off the results.

I understand Corts statements about Scientists being unwilling to push the boat out, using other methods other than PCR when the science study detected positives with just PCR alone. And i belive its right for him ( us ) to address that.

I dont think thats a good enough reason not to use the other methods in the science study, especially as we have learned of some pitfulls in storage, handling, and even PCR machines giving different results ( Hanson ) under these observations, it seems rather short sighted not to broaden the methods. If any ( or other problems yet to be discovered ? ) could be at the heart of most, or some of the discrepent results so far. As i think is being suggested by Asleep.

To ignore these possible pitfulls does seem a little odd i have to agree. Even though they may consider the detections by the WPI using PCR alone as evidence, that indeed, that is all that is required ?

But as has been mentioned, one PCR study may not be identical to another PCR study, possibly for a mryiad of reasons. some mentioned here, and possibly some unkown as of yet ? So i do think we all agree this needs to change. And i belive a message needs to be sent around the world of our concerns that one PCR experiment may not be replicating another, and point out the possible reasons we know about ( and possibly ones we do not, as of yet ) And urge any more researchers to not do PCR alone, regardless that the WPI seemed to do so well. Its no indication that wpi didnt fall into a as yet, unknown problem, throwing off the results, of other PCR experiments.

it may seem unlikely agreed. But other unlikley things have happened in science before. The stakes are too high in this, and the truth to important to everyone, patient and healthy alike. To disregard the possibileties. However unlikely they seem, Especially as the Science study itself is so compelling. Should be reason enough to be very very catiouse. One last thing it again does seem so very odd that VIDPX seems to get detections almost easily, Correct me if im wrong Norway, Cheney. and many private patient detections. but the rest of the world seems to struggle. Its almost as if all we really need to do is send them to VIDPX and hey presto, we will get more positive study results. ( im aware of the non detecttion recently of spinal fluid ) What gives with that observation, i cant be the only one to notice that discrepency. Isnt it why any studys now showing xmrv positive detections done by VIDPX are not considered valid evidence anymore. Again what gives with all this ? No evidence that VIDPX cant be trusted. But yet there science is now invalid to many reserachers, because they are not detecting xmrv. the way VIDPX is

So one asks why the Hell does VIDPX seem to be doing so, so easily ? Just like the WPI ?

It almost does feel like a conspiracy ( im not saying it is ) . or a major problem with either of those groups. Or as mentioned the only other third possible alternative. A as yet uknown unproven anomaly. or combination of anomalies, affecting detections around the world. The answer im convinced is in one of those three. so which is it guys place your bets

 

[jace]

This is the second Spanish study. The first was a small one out of Can Ruti Hospital, Barcelona, with around 15 patients. It was funded with help from patients, and Mikovits showed them the techniques necessary. It was positive, they found XMRV in similar percentages to the other positive studies, but the study group are not able to access further funds to continue their work. This study has failed to find a publisher so far, as has the WPI UK Study.

This second one was publicly funded. Like all the other negative studies we have seen, finding publication presented no problems.

One must ask why it is that so many positive studies have difficulty finding publication, while negative studies get straight into print, sometimes as little as a few days after submission. Don’t say it is down to the quality of the studies involved, because we have seen how very poor the negative studies have been. Unless poor study design is a prerequisite for publication

Remember the CDC and the Lo/Alter studies, last summer?

Back to this latest Spanish study. The thermodynamic task of locating a viral sequence integrated in DNA and merely mixed with DNA is very different and the parameters involved in the chemical reaction have to be changed accordingly. This needs DNA from a known clinically positive sample. As we know, the WPI offer to supply a known positive sample.

The technique of calibrating PCR conditions to locate a synthetic clone simply does not work and would defy the known laws of thermodynamics.

Continuing to use a technique with a 100% failure rate is beyond incompetent. Good scientists copy successful techniques, not unsuccessful ones. The people who failed to find the virus all used variations of the same technique which has always failed. Why test for XMRV using techniques which are now known not to work?

Again, they calibrate with an artificial clone as have all the other negative studies.

Danielson, 2010 and Klimas 2010 both have laid out clearly the problems of finding XMRV and it’s family of HGRV’s. Yet so many study designs fail to take notice.

 

[Sam Carter]

...

I stand corrected on the 67%. But to repeat what I've said elsewhere, the issue is not whether XMRV can be found by PCR (it can), but instead whether it can reliably detect wild-type PCR under unvalidated conditions (it cannot).

...

The WPI was able to detect "wild-type" XMRV using an unvalidated PCR assay.

The Huber-laboratory was able to detect wild-type XMRV and (wild-type?) MLV-like sequences using, by your metric, an unvalidated assay.

So what gives?

Who is the honest scientist?

 

[Cort]

The WPI was able to detect "wild-type" XMRV using an unvalidated PCR assay.

The Huber-laboratory was able to detect wild-type XMRV and (wild-type?) MLV-like sequences using, by your metric, an unvalidated assay.

This is something I never thought of until recently. Yes, wild-type virus would obviously be better - and as I noted I have been told that upcoming studies will be designing their tests using it but nobody had isolated the virus before the WPI I don't believe and they found it using what they had - which I assume was Silverman's clone...So unless I have something wrong it can be found - and in apparent abundance (???) using that at least in those patients....(which is why we need good patient cohorts) Maybe someone with more experience can show differently.

 

[Forbin]

From the addendum to the Science paper...[emphases mine]

In our October 2009 publication, we established XMRV infection in the blood products of our patient population by five different methods. Of these methods, single-round PCR on DNA from peripheral blood mononuclear cells (PBMCs), the least sensitive method, required us to use samples from a subset of chronically ill patients we had observed to have persistent viremia. In Figure 1A of our Science paper, we showed that DNA of 7 of 11 patients exhibited the expected gag and env PCR amplification products from single-round PCR with XMRV primers. We included this figure to demonstrate that nested PCR, which inevitably raises questions of contamination, is not essential to detect XMRV in highly viremic ME/CFS patients. The remaining 90 samples described in the paper exhibited very few XMRV-gag specific PCR products and no env specific PCR products following single round DNA PCR of DNA of unstimulated PBMCs.

What I take from that is that, if you want to find XMRV via single-round PCR on DNA, the samples need to be from ME/CFS patients who have tested positively for persistently high levels of infection with other viruses.

 

[Snow Leopard]

So one asks why the Hell does VIDPX seem to be doing so, so easily ? Just like the WPI ?

The WPI/VIPDx techniques were originally heavily based on "what worked" from the NCI methods. An argument can be made that they have the most experience with XMRV.

That said, only ~40-60% people are testing positive on the VIPDx tests, that range includes the weird results where people are only positive on a single method eg negative on culture, positive on serology.

But an argument can also be made that their tests have high false positive rates. We just don't know until there is more standardisation and correlation of the results between different labs and different methods.

 

[Cort]

In our October 2009 publication, we established XMRV infection in the blood products of our patient population by five different methods. Of these methods, single-round PCR on DNA from peripheral blood mononuclear cells (PBMCs), the least sensitive method, required us to use samples from a subset of chronically ill patients we had observed to have persistent viremia. In Figure 1A of our Science paper, we showed that DNA of 7 of 11 patients exhibited the expected gag and env PCR amplification products from single-round PCR with XMRV primers. We included this figure to demonstrate that nested PCR, which inevitably raises questions of contamination, is not essential to detect XMRV in highly viremic ME/CFS patients. The remaining 90 samples described in the paper exhibited very few XMRV-gag specific PCR products and no env specific PCR products following single round DNA PCR of DNA of unstimulated PBMCs.

Ha! That's interesting. I agree - if you're going to use single round you'd better do it on highly viremic patients. I think, though, that doesn't apply to most studies? Most studies are using nested PCR to find XMRV - as the WPI - are they not?