XMRV Buzz: WPI Responds and Dr. Singh Talks (May 16th)

May 16, 2011

Posted by Cort Johnson

WPI Response to the Singh Study

The WPI’s first response to the negative XMRV CFS Singh paper didn’t take long to come out. (Apparently a longer response will appear in the Journal of Retrovirology).

Hiding in Plain Sight? WPI Refutes Contamination Theory – Claims Distinct XMRV is Present – For over a year the WPI has gone along with the conclusion that their published samples from ME/CFS patients were essentially identical to the prostate cancer samples. The Science paper stated that the ‘complete XMRV sequences are >99% identical…to those detected in prostate cancer.

Now the WPI may be stating that one of the CFS samples they deposited in GenBank is, in fact, distinct from the type of XMRV found in VP62 and the 22RV1 cell line.  Since they don’t appear to have added samples to GenBank recently (and in fact state that they are in the process of doing so) their statement seems to infer that this strain is already in GenBank. If that’s true then their claim that this sample is actually genetically distinct from the others is a change of position that puts them at odds with other researchers.

Coffin Disagrees – Hue’s genetic analysis indicated that the WPI’s strains were not distinct from VP62/22RV1 and in  an LA Times article Dr. Coffin disagreed with their conclusions stating the differences amount to a few base pairs in 8,000. “That’s about the amount of difference I would expect to accumulate during one or two tissue culture passages,” wrote Tufts University retrovirologist John Coffin.

It Really Will Take a Family – The problem, of course, is that any gene sequence that appears to be associated with 22RV1 is now suspected  (but not proved) of being a contaminant. If the WPI can prove that they have distinctive strains of XMRV that have been altered by their passage through the human body they can put a big dent in the contamination theory.

Essentially what they want to do is establish the presence of a variable XMRV family that includes the pMLV sequences both they and Dr. Lo have found as well  as the VP62 XMRV, other ‘similar’ types of XMRV, the p forms (pMLV’s found by Lo et. al.) and other related human gamma retroviruses. The CDC’s recent finding of distinct strains of XMRV in prostate cancer cells does suggest that these strains are out there…but are they in CFS patients?

XMRV Sequences From the WPI Going Into GenBank (finally) – The multiple XMRV sequences  they have submitted to GenBank will help to answer that. The WPI has been stating for over a year that they have proof of more variability but for whatever reason have never put the sequences them into the GenBank repository for other researchers to study.   Now they are and the arguments can (and have already begun) on what constitutes a ‘distinct’ sequence. Its clear that the WPI will need more ‘distinctness’ in the GenBank to convince Dr. Coffin and others that they’ve found something really new.

The WPI also stated that they have extensive controls in place to prevent contamination and that about a third of the 3,000 tests they have done have been positive for XMRV.

Dr Singh Talks

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. al’s pMLV’s)  and other strains and that Dr. Singh’s use of an XMRV  clone developed from the VP62 strain lead her to miss those strains and that is one reason that she didn’t 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 WPI’s argument regarding using a human isolate possibly explain reduced results  but it’s 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.

 

 

 

 

20 comments

{ 20 comments… read them below or add one }

Ronnie May 16, 2011 at 11:52 pm

“Note though that the WPI did say that some of the samples provided to Dr. Singh tested positive for antibodies to the VP62 strain”

I know WPI and Dr. Singh have tested _different samples_ from the _same person_. But did WPI really test the _same sample_ as Dr. Singh?

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Cort May 17, 2011 at 7:35 am

No, I believe a phlebotomist took new samples from the WPI positive patients; thus they were testing different samples from the same people. That is the method being used in the BWG study.

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Ronnie May 17, 2011 at 11:40 am

Yes, that is my understanding also. But isn’t the quote from the post saying the opposite?

“Note though that the WPI did say that some of the samples provided to Dr. Singh tested positive for antibodies to the VP62 strain”

Shane May 17, 2011 at 8:34 am

Did you ask her how it was that she found no XMRV in the blood of any of the 100 CFS patients or 200 controls and yet she maintains that her prostate tissue study found 27% in patients and 4% in controls?

Forgetting about whether or not her results support the WPI’s findings – taken together, don’t the results of her two studies raise questions about the ability to find XMRV in blood (of complex, living breathing humans). Does she feel that it is even possible to find XMRV in the blood and if so, why didn’t she find roughly 4% in both CFS patients and controls? If not, then what does she think her study contributes to our understanding of XMRV, CFS, science?

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Cort May 17, 2011 at 1:24 pm

No I didn’t. I think there are numerous unresolved questions about XMRV’s presence in every tissue. Dr. Singh appears to believe her immunohistochemistry results are solid but they have been challenged by two studies. The prostate findings could be spurious – altho the recent CDC study suggests that distinctive infective isolates have been found. It’s also possible that XMRV is in prostate tissues and is not showing up in the blood.

I don’t think there’s any question about her or other labs ability to detect XMRV in the blood. The BWG found that all the labs in the study (five then, dropping down to three now), can detect small amounts of XMRV in the blood. Her labs and others test their ability to do so using VP62 or other isolates. While VP62 may differ from other strains of XMRV – that isolate worked very well in the Lombardi et al study – and while it might not pick up all strains of XMRV – it should still be able to pick some.

As Ronnie noted, the WPI reported that some of the patients they directed Dr. Singh to tested positive for antibodies to the VP 62 strain. At some point she should have gotten positives from these patients. The fact that the WPI directed her to patients testing positive for the VP 62 strain suggests that a significant of people who have tested positive for XMRV test at the WPI test positive for the VP 62 strain.

The 4% question was a good one – it was covered by Mindy Kitei in her interview with Dr. Singh. It’s asking that two very different tests in very different tissues have similiar results and that might be asking a bit much at this point. I don’t think that issue reflects on Dr. Singhs ability to replicate the Lombardi et al papers methods or on her ability to detect XMRV in the blood.

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Simon May 17, 2011 at 11:16 am

The sequences are in the GenBank.

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Omer May 17, 2011 at 11:39 am

Cort, I think you are mistaken and therefore misleading others: I don’t think that the WPI is saying that “you need to use a natural isolate”. What they are saying is the following: We have positive samples. You should calibarate your assays against our samples, until you find all of them positive. Than you know that your assay is capable of detecting XMRV that lived in the blood of that patient, and that might be somewhat different than VP62. In the original “Science” study the WPI sequenced 2 full viruses and one partial sequence. Since then, they found that there is more diversion to the sequences. Therefore, if I understand correctly (and perhaps I don’t), what they say is as follows:
1) Your assay must be an assay that would setect a large scale of sequences that are close enough, and we have samples that contain a large scale of sequences.
2) Your assay must be able to detect viruses that are in the blood after infecting the human being, not after you created them in the lab and put them in a sample. I think that Dr. Singh also said such a thing before (talked about matrix in body, or something, I don’t really remember, but it was about that, I think).

You should correct your mistake, I believe, unless you’d contact the WPI and they will tell you that you weren’t mistaking in telling us what they are saying (and, as I said, I think that they will have important corrections to what you said).

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Cort May 17, 2011 at 1:38 pm

You have a point Omer. I think the WPI is saying that Dr. Singh should have used a ‘natural isolate’ and since they are the only ones who have found XMRV in CFS patients to this point – they are the only ones who could provide that.

I think some of your points would be well taken if Dr. Singh had found 10 or 20 or 30% of the patient samples were positive and it was a matter of finding all of the XMRV strains present but that unfortunately was not the case. Remember that the VP 62 clone was used to validate the Lombardi papers results and it was effective in picking up XMRV in that set of patients. That makes it difficult to believe that using that clone now shouldn’t at least pick a significant amount of the XMRV present.

Remember also that the VP62 clone produces infectious XMRV that is essentially identical to the sequences the WPI deposited. Yes there were only 2 1/2 sequences but the odds of plucking out 2 1/2 sequences which were utterly distinct from the rest of the samples must be astronimical. Plus the original paper also stated thatall the env sequences, I think they were, were also identical to tholse found in prostate cancer samples. This suggests that the rest of the samples may genetically be very close to the VP62.

The WPI has submitted more samples to GenBank and, since genetic variation is a key component of the contamination theory, an analysis of those will be very important – altho already we have some controversy over what the WPI considers ‘distinct’ and what others do…..Nothing has been simple with XMRV :) .

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John May 18, 2011 at 2:53 am

It’s not only the WPI who just happened to pick out XMRV isolates with nearly identical sequences, the Cleveland Clinic also did the same because it was their sequences that the WPI’s were identical to!

Also, glad to see that the WPI’s unofficial grace period appears to be coming to an end. Based on Dr. Singh’s “Any reputable lab that uses PCR as a test on clinical samples, uses dUTP-UNG… I do not know why Lo et al or Lombardi et al did not” comment and Dr. Stoye’s comments in the Tribune, it looks like the retroviral community has had just about enough of being called incompetant and/or liars by the WPI and it’s followers.

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Cort May 18, 2011 at 2:09 pm

Here are Dr. Satterfield’s comments on Dr. Singhs response

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 PCR’s 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 (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.”

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 billions 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.

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Megan May 18, 2011 at 6:45 am

Cort,

Did you ask Singh what she thought her positive antibody tests were picking up in CFS patients – the antibody tests she referred to a couple of times in her patent document? If not XMRV, then what?

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Cort May 18, 2011 at 2:07 pm

Didn’t think to think of that Megan…I think she did do antibody tests in this study but I do remember a set of antibody tests in the patent…As I remember she thought she had identified ones that worked and ones that didn’t work so.

The patent application was certainly a source of hope. I don’t know if she’s using different antibody tests or not. I guess we could go back and look and see…

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LJS May 18, 2011 at 10:52 am

Sorry to nit pick but the title of this article is incorrect, it says “(Apr. 16th)” : )

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Cort May 18, 2011 at 2:04 pm

Not at all – thanks for telling me :)

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Megan May 18, 2011 at 8:27 pm

Cort,

Below are the relavent quotes from Singh’s patent document. I understand that both of the below references refer to antibody tests, which obviously could not be affected by contamination. I hope if you (or anyone else) gets to speak to her again that this question is asked, as it seems most pertinent.

[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.

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John May 19, 2011 at 2:59 am

There is a common refrain that ‘contaminants don’t cause an immune response’, but I think one important thing to remember is that not all patients that are even reported as being ‘XMRV +’ have antibodies either. As far as I know the WPI has never made a complete report on the concordance of their results, which would be an important step due to some patients testing positive by PCR but not antibodies/culture, some will test positive by culture but not antibodies/PCR and some will test positive by antibodies but not PCR/culture. I also think I remember reading that none of the controls in the Science paper that tested positive for XMRV had antibodies, so according to the above logic that ‘contaminants don’t produce an immune response’, then apparently neither does XMRV infection!

Also the Emory monkey study developed antibodies that were highly specific for XMRV and tested over 1,000 blood donors for these antibodies. Results? No positives.

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Megan May 19, 2011 at 5:55 am

John,

Thanks for your response, but my question was not about the concordance of the WPI’s test results, it was about Singh’s report of positive antibody responses in CFS patients that were apparently not in controls. To me this whole debate is not about ‘who’s right’ about XMRV, it’s about the association of a virus (XMRV or something else) with CFS.

The XMRV test referred to by Singh in her patent document is obviously not as specific as that used in the monkey study, as you have pointed out, or she would have reported a positive result in her CFS study. That’s precisely the point I was trying to make in the post – is there something else here (perhaps similar but not XMRV?) that was getting picked up in those tests?

progestin side effects May 22, 2011 at 2:16 am

I don’t think there’s any question about her or other labs ability to detect XMRV in the blood. The BWG found that all the labs in the study (five then, dropping down to three now), can detect small amounts of XMRV in the blood.

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John May 23, 2011 at 9:50 am

One thing that has been kind of overlooked in some quarters in this whole thing is that the Singh study not only could but actually did find XMRV. They found it in equal amounts in the patient and control samples and traced it back to the robot they used to extract DNA in a previous study. When they quit using the robot the contamination stopped occuring. In fact the fact that they found such minute amounts gave them even more confidence that they should have been able to detect XMRV in patient and/or control samples should those samples actually have contained any XMRV. It kind of brings to mind Dr. Hanson’s comment about using different PCR machines provides different results, no?

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Cort May 17, 2011 at 1:50 pm

That quote does say the opposite! It was a mistake :( and I have fixed it. Thanks for pointing that out. The WPI did not directly provide samples to Dr. Singh. They provided patients and Dr. Singh used a phlebotomy service to collect their blood.

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