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XMRV - The case against contamination

Blog entry posted by Bob, Jul 2, 2011.

Feedback, corrections, discussion and criticism are very welcome.

I've also attached a PDF version for easy printing.

Contents

  • Preface
  • Introduction
  • Contamination theories
  • The WPIs methodologies
  • Lack of sequence variation
  • John Coffins conclusions regarding the origins of XMRV (A recombination event)
  • Switzer - Publishes new XMRV varieties, and challenges Coffin's recombination theory
  • Conflicting statements and conclusions from scientists promoting contamination theories
  • Other arguments against contamination
  • Ongoing Research
  • Some of the scientists finding XMRV
  • References

Preface

Ive collected together some of my posts from the forum about XMRV and contamination.
The numbers in brackets, e.g. (1), point you towards the 'references' at the bottom of the page, for further info.

In this blog, I discuss the evidence that I believe demonstrates that each of the contamination theories relating to XMRV is not based on conclusive evidence, or even based on the balance of evidence.

With such an overwhelming number of negative XMRV/CFS studies published, it is easy to assume that XMRV in CFS patients is just a case of contamination, and that the WPI's research is just plain wrong.

I am not trying to argue that contamination isnt an issue to be taken seriously, but I am presenting the evidence to show that the case for contamination has not been proven, and that there is still a lot more research to be carried out before we gain a good knowledge about XMRV, and any related viruses.

I've avoided detailed analyses of the specifics of individual studies, but I've provided links for further information, in the 'references' section.

I'm not going to cover all the issues, but I'm going to try to explain why our total knowledge about XMRV is still in its infancy and why it is far from proven that the XMRV discovered in CFS patients is due to contamination.


Introduction

Although there have now been a large number of negative XMRV studies, there has also been a handful of positive XMRV studies (14), mostly unrelated to CFS. On top of this, there are a large number of ongoing investigative XMRV studies (14), which, for example, look at the cellular action of XMRV. There is still only one published positive study that shows a convincing association between XMRV and CFS, but there are reportedly a number of positive XMRV/CFS studies in the pipeline (15).

The Alter & Lo study which detected P-type MLV-related viruses hasn't satisfied everyone, in terms of it being an XMRV validation study, but the results of the Alter/Lo study are interesting in their own right, and have probably given added impetus to the XMRV reseearch.

There is a great deal of ongoing scientific investigation into XMRV, and unfortunately it's probably going to be a long time before there are any firm conclusions about if, and how, XMRV affects ME/CFS patients and the rest of the population.

It has been stated by various people, that XMRV is a contaminant, or that XMRV is not a real virus, or that there is no association between XMRV and CFS/ME, or that XMRV is not a human virus. The CFS community has also been told that its a bust, and that we should forget about XMRV and not worry ourselves about it.

But all of these statements and conclusions are currently just opinions because there is no scientific consensus, as the many published research papers offer conflicting results and evidence. There is so much that we don't know about XMRV that it is far too early to draw any conclusions from the relatively tiny amount of research that has been carried out so far.


Contamination theories

These are the contamination theories that we have to date:

Theory 1. Detection of XMRV is due to false PCR readings (i.e. cross reactivity).
Theory 2. XMRV is not a virus but only mouse DNA contamination.
Theory 3. XMRV is purely a mouse virus.
Theory 4. XMRV is DNA contamination from a cell line.
Theory 5. XMRV is purely a cell line virus (Purely a lab artifact).
Theory 6. XMRV is a wild human virus but doesn't exist in CFS patients.

The WPIs research and the original Science paper have been accused of all of these.

There seems to be a general scientific consensus that the first 4 have now been disproved, and that XMRV is actually a real virus. That doesnt make the WPIs research immune from the first 4 contamination theories, but the strength of the WPIs original Science paper is such it is hard for these theories to stick.

So lets go through each of these theories in turn...

Theory 1 - Detection of XMRV is due to false PCR readings (i.e. cross reactivity):
The WPI have isolated and sequenced their sequences, and so we know exactly what they detected.

Theory 2 - XMRV is not a virus but only mouse DNA contamination:
The WPI have isolated and sequenced what they detected, and there seems to be a consensus that they have indeed sequenced a virus called XMRV.

Theory 3 - XMRV is purely a mouse virus:
There have been a number of studies that have now detected XMRV in humans, including the prostate cancer studies, and a study by William Switzer of the CDC who concluded in a recently published paper (1) that XMRV is a human virus. John Coffin has a different theory - that XMRV is not a human virus, and not a mouse virus, but that it is purely the result of a recombination of two mouse virus in a laboratory cell line.
XMRV has not been detected in any mouse to date.

Theory 4 - XMRV is DNA contamination from a cell line:
This is similar to the theory that XMRV is only mouse DNA contamination, but the contamination comes from a cell line rather than from lab mice. Since researchers have detected XMRV, as a whole virus, in prostate cancer cell lines, the contamination theories have moved onto theories 5 and 6...

Theory 5 - XMRV is purely a cell line virus (Purely a lab artifact):
The latest theory, from John Coffin & his colleagues, in a published paper (Paprotka et al.) (16), is that XMRV is a real virus, but that it only exists in a prostate cancer cell line, as a 'laboratory artifact'. Coffin and his colleagues have concluded that XMRV is a real virus, but that it has never existed outside a laboratory, and it has never infected humans, therefore all positive XMRV research can only have detected lab contamination. These conclusions are partly based on a lack of variation in the WPI's original published XMRV sequences, when compared with the sequences from the original prostate cancer study. Coffin says that this lack of genetic variation demonstrates that XMRV cannot be a human infection. And the conclusions are also based on Coffin and his colleagues finding two endogenous mouse viruses, which they call pre-XMRV1 & pre-XMRV2, in the mice onto which the prostate cancer cell line, that they were investigating, was grafted. Coffin and his colleagues conclude, in their published paper, that sections of DNA of each of the two pre-XMRV viruses are almost identical to the originally published XMRV sequences and that the pre-XMRV viruses simply 'recombined' to create XMRV in the prostate cancer cell line.

Coffin's study checked past versions of the prostate cancer cell-line to find out when XMRV entered, or was created in, the cell line. They concluded that XMRV was created in a lab, in the cell line, in the mid-1990's, and that there is a 'trillion to one' chance (i.e. highly unlikely) of it being created at any other time or in any other place. The Paprotka et al. study also brings the original Urisman et al. prostate cancer study (24) into doubt, as the suggestion is that Urisman et al. also only detected a laboratory artifact, and not a human infection.

However, other prominent scientists do not subscribe to 'Theory 5' because there have been a number of studies that have convincingly detected XMRV in human tissue and blood (1) (3) (11), and all of the prominent studies are still backed by their authors. So one of the weaknesses of Theory 5, is the evidence provided by other scientists such as Dr Singh (17) and William Switzer (1), which suggests and demonstrates human infection. Mikovits says she continuously checks for contamination at all stages of her procedures.

But alongside this, a recently published study by Switzer (1), and two abstracts from Switzer (8) (9) demonstrate XMRV genetic variability (1) and also challenge Coffin's recombination theory (8) (9), thus opening up the possibility that XMRV originated in other places, other than the cell line that Coffin investigated. So this evidence demonstrates further weaknesses in Coffin's and his colleagues' conclusions.

Switzer concludes that XMRV is indeed a real human virus, with genetic variation to match human infection, and that XMRV could have originated in a myriad of different MLV recombination events. (See more information about this below.)

Theory 6 - XMRV is a wild human virus but doesn't exist in CFS patients:
Singh's theory is slightly different to Coffin's because, when she is referring to her prostate cancer study (3), she concludes that XMRV exists in the human population, in prostate cancer patients at 23%, and in the normal healthy population at 4% (or 6%?). But when Singh is referring to her CFS study, where she tested blood instead of tissue, and where she failed to detect XMRV in any blood samples, she concludes that XMRV does not exist in CFS patients or in the normal healthy population (0%). The weakness of Singhs conclusions is that they are conflicting, based on opposing results using different methodologies. In her CFS study, XMRV exists in 0% of the normal population, but in her prostate cancer study, XMRV exists in 4% (or 6%?) of the normal population. So, Singh apparently subscribes to theory 6, although in a rather contradictory fashion, as her prostate cancer study suggests that at least 4% of CFS/ME patients should test positive for XMRV. It seems to me that Singh has merely demonstrated that she can detect XMRV in tissue, but not in blood samples. She has no other explanation for the discrepancies in her results (4). (See more about the Singh studies below.)

Switzer also believes that XMRV is a real wild human virus (1), but that it doesn't exist in CFS patients, and that it is not associated with prostate cancer, so he also subscribes to theory 6. However, his opinions are also contradictory, because he admits in a recent study (1) that he uses methodologies inadequate for detecting XMRV in the blood of any individuals, even when he has confirmed them to be XMRV-positive by testing prostate cancer tissue from those individuals.

So the weakness of theory 6 is the fact that the scientists who have published some of the zero/zero XMRV studies, looking at the blood of CFS patients, have demonstrated that they are unable to detect XMRV in the blood of XMRV-positive individuals. This gives us a good reason to suspect that it might be the methodologies that are at issue, rather than an absence of XMRV. There might be other reasons for the discrepancies, but we just don't know the exact reasons at the moment, and nor do the scientists.

The other weakness of theory 6 is the consistent results from the WPI, and the strength of all of their various findings. Then there are the related MLV sequences that Alter and Lo detected. These findings suggest that further research into human infection from MLV-related viruses is worth continuing with.


In terms of the specific allegation that the WPIs results are purely due to contamination, this has been argued over endlessly, so I will just repeat the following...
A lack of sequence variability is not proof of contamination for the following reasons:
1. XMRV Mutation behaviour could be similar to HTLV. (6)
2. There could be alternative routes of XMRV transmission in humans such as in vaccines. (5)
3. The WPI have many more XMRV isolates that they have been unable to sequence and publish due to a lack of funding. As our knowledge about XMRV, and the evidence relating to XMRV, continues to grow, then a higher variation in genetic sequences might become evident.


There are separate contamination issues that should be clearly distinguished when talking about contamination in relation to the WPIs research. Whatever the contamination outcome in any individual research study, it will not determine the nature of XMRV. So, for example, if the WPIs research results are proven to be due to contamination, this does not mean that XMRV is not a real human virus. If the WPI have detected the XMRV virus in their research, it could still be a contaminant from either a lab or human source. Equally, if the WPI have not actually detected XMRV in their research, this does not automatically disqualify XMRV from being a real human virus.
The contamination issues need to be separated in any discussion. So, in other words, we need to keep in mind that XMRV may or may not be a real human virus, whatever the contamination status of the WPIs research.

Here are some virologists who believe that XMRV is a real virus (and I'm sure that there are more):
Switzer (1), Alter (7), Russceti (14), Beiger (15), Hanson (12), Silverman (2), Singh (17), Lombardi.

Harvey Alter strongly believes that the WPI's research is evidence of a real human virus in CFS patients (7) (13). It has been said that Alter has weakened the strength of his belief about the XMRV research, but there is no evidence for this as yet, and he has recently said that any current XMRV contamination theories do not affect his own PMRV research results.

Switzer has provided his own XMRV sequences to genbank (1) (18) which he concludes is evidence that XMRV is a real, wild, human virus, due to the genetic variability of his sequences.

Scientists like John Coffin and his colleagues, believe that XMRV is a real virus, but that it only exists in laboratories, and has not entered into the human population.


The WPIs methodologies

Judy Mikovits has said that PCR is the least effective method to detect XMRV. In the addendum to the Science paper (19), the authors state clearly that they used 5 different methods, and that single-round PCR was the least sensitive of the methods used, possibly because of the very low copy numbers of the virus in the blood. So the authors selected just a small cohort of 11 patients to test using this method, who they had observed to have persistent viremia (viremia = virus in the blood). 7 out of the 11 tested positive for gag and env XMRV sequences.

"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."

"In contrast, when cDNA was prepared from PBMCs, 67% of the samples exhibited gag products upon nested PCR, though PCR with nested env primers did not result in detectable products from these samples."

"Of the 34 patients whose PBMCs were negative for XMRV by DNA or cDNA PCR, 17 were positive for infectious virus when co-cultured with the LNCaP indicator cell line, as XMRV gag and env PCR products were detected in the cell line following their infection with XMRV from the patient PBMCs"

So when the authors of the negative (zero/zero) studies found that they couldnt detect XMRV by PCR, then it was not a surprise. Judy Mikovits and her co-authors couldn't either, except for 7 out of the 11 carefully selected patients who were probably extremely ill, and showing specific signs of viremina. (I don't know the details about the signs.)

It's only by using further techniques, such as nested PCR, stimulated PBMCs, and by culturing the samples, that Judy was able to detect XMRV in most patients.

When the authors of the negative studies say that they did exactly what Judy did, her methodology was very complex and they haven't followed it exactly, and some not even closely.

Here's what the authors of the addendum say about the UK zero/zero studies: "...very few, if any, of the samples would be expected to be positive by DNA PCR..."

These are the results from the Science paper:
Unstimulated single round PCR (gag) detected 7% positive XMRV DNA.
Unstimulated nested PCR (gag) detected 21% DNA and 54% cDNA.
Stimulated nested PCR (gag) detected 72% cDNA.
Cultured LNCaP nested (gag) detected 89% cDNA.


Lack of sequence variation

Paprotka et al. published a paper (16), that concluded that the XMRV DNA sequences published, in relation to the Lombardi et al. Science paper and the Urisman et al. prostate cancer study, do not have enough variation or mutation for them to be a human infection.

But there are a number of reasons why this theory is not solid, and there are a number of reasons why any lack of variability in the WPIs published viral sequences does not prove contamination.

A letter published in the Lancet (5) discusses how any lack of genetic variation could indicate another route of transmission for the virus into the human population, rather than from human to human. If the virus originated in a laboratory, then people could be infected directly from the source of contamination, such as, for example, via vaccines.

Also, it seems that HTLV, a human retrovirus, also has a very low rate of mutation (20)(21)(22), and so XMRV could have a similar behaviour to HTLV.

The WPI have recently published (18) new sequences which have not yet been subjected to a peer reviewed analysis, as far as I am aware.

However, in a separate study on prostate cancer patients, William Switzer has now published sequences from 3 XMRV isolates (1)(18), which he concludes display genetic variation such as would be expected in human infection. Switzers conclusions cant tell us anything directly about the WPIs published paper, but he does confirm that XMRV may indeed by a wild human infection.


John Coffins conclusions regarding the origins of XMRV (A recombination event)

John Coffin, and his colleagues (Paprotka et al.), have published a paper (Recombinant Origin of the Retrovirus XMRV) (16) which concludes that XMRV was created in a laboratory, after the recombination of two pre-XMRV mouse viruses, in a prostate cancer cell line. The authors conclude that there is a trillion to one chance that XMRV could not have been created any other way (i.e. almost impossible.) They also say that the lack of variability in the gene sequences published in the WPIs original study, and the original prostate cancer study by Urisman et al. (the Silverman paper), indicate that XMRV has not infected humans, but is only found in the cell line, and is therefore purely a lab artefact.

There are a number of reasons why Coffin's conclusions are premature:

1. A wider variety of XMRV sequences have now been detected and published in genbank, by William Switzer. Switzer concludes that the variation in his sequences indicate human infection. (1)(18)

2. The original prostate cancer sample, which initiated the cell line, may have harboured undetectable minuscule quantities of XMRV, which then flourished as the cell line was propagated, making it detectable only in later samples of the cell line.

3. Switzer's recent abstracts, regarding potential MLV recombination events, challenges Coffin's conclusions, because Switzer has worked out many other possible recombinations. Switzer's recent study challenges the Paprotka et al. study. (8)(9)

4. Coffin might have only detected contamination in his own study, in which case all of his results and conclusions are invalid.

5. The WPI say that they have many viruses that they can't afford to sequence.

But even if there is no significant variation, this does not confirm that it is contamination, for a couple of reasons:
1. A possible different route of infection, such as via vaccines.
2. A possible virus mutation pattern similar to HTLV.


Switzer - Publishes new XMRV varieties, and challenges Coffin's recombination theory

There have been a couple of studies (one paper & one abstract) recently published by William Switzer of the CDC, which have a significant impact on the XMRV contamination debate.

Here are details of the two studies...

Switzer study 1

In his recently published study, William Switzer of the CDC specifically says that the XMRV sequence variation that he has detected is "consistent with virus evolution during spread and persistence." Switzer's evidence and conclusions directly contradict Coffins and his co-authors conclusions in relation to there being no variation in any XMRV sequences. Switzer indicates there is evidence of virus evolution expected during normal human infection in the wild.

No Association of Xenotropic Murine Leukemia Virus-Related Viruses with Prostate Cancer
William M. Switzer, Hongwei Jia, HaoQiang Zheng, Shaohua Tang, Walid Heneine
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019065#abstract0

"Sequence analysis showed that patients 5935, 5956 and 6203 are infected with variant XMRV strains. The 168-bp pol sequences from all three patients showed 90.5100% nucleotide identity to each other, 94100% to XMRV, 91.798.8% to XMLV, 94100% to PMLV, and 91100% to ecotropic MLV (EMLV) in this short region.
164-bp env sequences from persons 5956 and 6203 were identical to each other and shared the highest nucleotide identity (94.9100%) to XMRV and other xenotropic MLV strains, respectively, [25]."
...
"The XMRV sequences from both prostate cancer patients were also distinct from the PMLV sequences amplified from the murine cell line (RAW) used for preparing WB antigens demonstrating further that these are not laboratory contaminants (Fig. 2). These results confirm the presence of XMRV in both patients and demonstrate that XMRV diversity is greater than currently appreciated."
...
"Sequence analysis of the PCR-positive specimens was highly informative because it confirmed that all three specimens were XMRV-related. Also, the finding of a viral strain in three prostate cancer patients that is distinct from the XMRV seen in previous studies is significant and demonstrates a broader viral diversity. This would be an expected result consistent with virus evolution during spread and persistence."


http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019065#abstract0

Although Switzer concluded that there is no association between XMRV and prostate cancer, based on the findings of this study, he did detect XMRV in the tissue of 3 prostate cancer samples, but he could not detect XMRV in the blood of those XMRV-positive patients. He concluded that further investigation of XMRV is needed.

"Sequence analysis of the PCR-positive specimens was highly informative because it confirmed that all three specimens were XMRV-related. Also, the finding of a viral strain in three prostate cancer patients that is distinct from the XMRV seen in previous studies is significant and demonstrates a broader viral diversity. This would be an expected result consistent with virus evolution during spread and persistence."

The significance of Switzer's study is...

1. Switzer confirms that XMRV is a real wild human virus.

2. Switzer concludes that the genetic variation is consistent with normal human infection.

3. Switzer admits that he can't detect XMRV in the blood of XMRV-positive prostate cancer patients, using established methodologies. This has relevance for all of the other negative XMRV studies.

4. Switzer confirms that his findings not due to contamination.

Switzer study 2 (Abstracts only)

The following two abstracts look like they are based on the same study...

Extensive Genetic Recombination in the XMRV Genome
William Switzer, W Heneine1, M Prosperi, and M Salemi
Presented at the 18th Conference on Retroviruses and Opportunistic Infections (CROI) 2011
http://retroconference.org/AbstractSearch/Default.aspx?Conf=20&Abs=40126

Murine leukemia viruses (MuLV) and Xenotropic MuLV-related viruses exhibit inter-tropic complex recombination patterns
Mattia C F Prosperi , William M Switzer, Walid Heneine and Marco Salemi
6 June 2011
Presented at the '15th International Conference on Human Retroviruses: HTLV and Related Viruses'
http://www.retrovirology.com/content/8/S1/A235

Extract from the Conclusion:
"Given the evidence of inter-tropic recombination in MuLV, detection and classification of recombination in XMRV using different MuLV tropism prototypes should be interpreted with caution ... These results suggest that identification of parental strains of the potential recombinants is difficult and that recombination in the highly genetically related MuLV have been occurring for some time."


The Paprotka et al. paper (i.e. Paprotka, Coffin and Pathak) (16) ruled out everything for XMRV other than a recombination event happening in the specific cell line that they studied. This CDC/Switzer study points out that, contrary to John Coffin's conclusions in the Paprotka et al. study, there are a myriad of ways that XMRV could potentially have been created by a recombination event, and that the prostate cancer cell line might just be one example of many possible recombination events. Switzer draws very different conclusions to Paprotka et al., and directly challenges their study's conclusion of a 'one in a trillion' chance of a recombination event.

The significance of this paper is that Switzer directly challenges, Coffin's theories about the recombination history of XMRV. Switzer appears to be saying that Coffin's 'one in a trillion' conclusion is mistaken, and that there is a vast potential for possible recombination events to have happened to create XMRV.

This study hasn't been published yet, so it is premature to make any definite conclusions from it, but taken together with the published Switzer study, above, the two studies challenge most aspects of the Paprotka et al. (e.g. Paprotka, Coffin and Pathak) recombination study.

Switzer's sequences published in genbank:

Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention.
Switzer,W.M., Jia,H., Zheng,H., Tang,S. and Heneine,W.
10th May 2011


Xenotropic MuLV-related virus isolate 5935 polymerase (pol) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HQ116790.1

Xenotropic MuLV-related virus isolate 5956 gag protein (gag) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HM003612.1

Xenotropic MuLV-related virus isolate 6203 envelope (env) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HM003611.1

Xenotropic MuLV-related virus isolate 5956 envelope (env) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HM003610.1

Xenotropic MuLV-related virus isolate 6203 polymerase (pol) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HM003609.1

Xenotropic MuLV-related virus isolate 5956 polymerase (pol) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HM003608.1


Conflicting statements and conclusions from scientists promoting contamination theories


Coffin:
So Coffin says: "...these results essentially close the door on XMRV as a cause of human disease."
But prostate cancer is still being investigated, as well as CFS, and breast cancer, and autism.
Switzer of the CDC has already challenged (9) Coffin's study with a new paper, which challenged Coffin's recombination theory:

Conclusion:
"Given the evidence of inter-tropic recombination in MuLV, detection and classification of recombination in XMRV using different MuLV tropism prototypes should be interpreted with caution ... These results suggest that identification of parental strains of the potential recombinants is difficult and that recombination in the highly genetically related MuLV have been occurring for some time."


This CDC study points out that, contrary to Paprotka's, Coffin's and Pathak's conclusions, there are a myriad of ways that XMRV could have been created, and that the prostate cancer cell line might just be one example of many possible recombination events. Switzer draws very different conclusions to Paprotka et al., and directly challenges their study's conclusion of a 'one in a trillion' chance of a recombination event.


Stoye:
"He said the implication was that the samples were contaminated, however this had not been definitively proven".
So it has not been proven that the WPI's results are due to contamination, but XMRV is 'definitely' not linked to CFS, according to Stoye!? This does seem rather contradictory, to say the least.

At least Stoye helpfully admits that contamination has not been proven.


Singh:

With regards to Dr Singh's research, she has made contradictory interpretations of her different published papers.

In her prostate cancer study, Dr Singh detected XMRV in 4% of the prostate tissue of healthy controls.
This confirmed and validated the WPI's research which detected XMRV in 3.7% of healthy controls.
But in Dr Singh's CFS study, she was unable to detect XMRV in the blood of any healthy controls (0%).

So, if Singh's prostate cancer study is valid, then it seems that she was simply unable to detect XMRV in any blood samples, possibly due to low copy numbers and inadequate techniques.

Instead of querying her own results, and asking herself if she was using adequate methodologies to detect extremelly low titre XMRV in blood samples, she simply concluded that XMRV is not associated with CFS, based on one single negative study.

Singh's study and abilities appear to be sophisticated, but if she was unable to detect XMRV in the blood using her methodologies, then maybe that is the only conclusion that she should draw from her results. (The WPI has listed the differences in the methodologies between the Lombardi et al. paper and Singhs paper.) (23)

When ask about the discrepancies, Dr Singh said that she didn't know why there was a difference:
"Not entirely sure, but there were different assays (e.g. immunohistochemistry) and different sample types (blood vs prostate tissue)."
http://www.cfscentral.com/2011/05/dr-ila-singh-we-are-now-convinced-that.html

So Dr Singh admits that the zero results maybe due to different methodologies.

But, despite this, she has remarkably come to exactly the same conclusions as Stoye and Coffin, as if they were all reading the same script:

"I'd urge people to move on rather than to keep their hopes hanging on the link between XMRV and CFS," says Ila Singh
http://news.sciencemag.org/scienceinsider/2011/05/more-bad-news-for-chronic-fatigue.html

Singh says about finding XMRV in prostate cancer patients:
"This increases the population at risk for XMRV infection from only those homozygous for the RNASEL variant, to all individuals. Our goal is to further understand pathogenesis by XMRV, to determine if this virus causes prostate cancer, and delineate possible mechanisms for oncogenesis. We will also carry out epidemiological studies to study the prevalence and tropism of this new and important virus."
http://projectreporter.nih.gov/project_info_description.cfm?aid=7866444&icde=4780366

So if XMRV is such a serious virus that deserves such close attention, then why dismiss the association with CFS based on the questionable results of just one study?

HIV research had a very similar pattern of unexpected research outcomes in the early years.


Other arguments against contamination

1. Alter and Lo replicated the WPI's research. They found a closely related MLV-like virus, which they consider to be a wild human viral infection. At the very least, this should give us a reason to continue the research into MLV-related viruses.

2. The WPI have, at the very least, found a biomarker, even if it were to turn out that they haven't detected XMRV. The anti-bodies that they have detected in ME patients don't exist in many normal controls. This seems like a huge step forwards for ME research in itself, whatever the outcome of XMRV.

3. The XMRV research is ongoing. There are many researchers who are investigating it, but we only hear about the most vocal ones.

4. XMRV is almost as elusive in prostate cancer patients as it is in ME patients, but there have been at least four positive prostate cancer studies which confirm that XMRV is a human virus, and at least a couple of unpublished studies (15). Just as in ME, not all researchers can detect XMRV in prostate cancer. This gives us extra information about the virus, as it may support the theory that XMRV exists in extremely low copy numbers, or is undetectable using established methodologies for some other reason.

5. There are a whole load of researchers who are treating XMRV as a human virus. It isn't just Judy Mikovits. Even the CDC is treating it as a human virus now. (see list, below.)

6. Switzer of the CDC detected a small number of XMRV positive samples in a prostate cancer study that he carried out. Although he concluded that this was a 'negative' prostate cancer study, he did confirm that XMRV is a human virus. He also confirmed that he could not detect XMRV in the blood of the XMRV-positive patients, because, he suggested, of the 'low copy numbers'. In other words, he has admitted that established PCR techniques are not adequate to detect XMRV in the blood, just as Judy Mikovits predicted people wouldn't be able to, using standard methodologies, especially single-pass PCR.

7. The theories that XMRV is just a contaminant have been continuously challenged by the evolving research. Ever since the Science study was published, the contamination theories have constantly changed to keep up with the evolving science. The latest Coffin paper to be published re the combination event, has now been challenged by both the CDC's latest paper, and by the new sequences published in genbank by both Switzer and possibly by the WPI.

8. Even if XMRV is a lab creation, that doesn't mean it isn't also a wild human virus. Even Switzer of the CDC now says it is a human virus.

9. There is plenty of research evidence to show that XMRV is viable in humans and human cells.

10. As far as I am aware, no one has yet specifically found XMRV contaminating any equipment, or reagents, anywhere. Only MLV's or MLV-like sequences, as far as I'm aware.

11. Mikovits and Lo have carried out contamination tests in their research.

12. The strength of the results of the original Lombardi et al. research, and the WPIs reported ongoing research, such as the UK study, suggests that they are not finding contamination.


Ongoing Research

XMRV research is being carried out by a large number of scientists right now, and having followed the research closely, I know that there is far more to the XMRV story than the high profile negative studies. If you are interested in seeing some of the ongoing research, then please have a look at these abstracts from a recent retrovirology conference:
http://forums.phoenixrising.me/show...ce on Human Retrovirology - List of Abstracts


Unpublished XMRV studies and Future XMRV studies:
http://phoenixrising.me/forums/showthread.php?12565-Unpublished-XMRV-studies-and-Future-XMRV-studies


Some of the scientists finding XMRV

Lombardi et al. (CFS)
http://www.sciencemag.org/content/326/5952/585.abstract

Alter/Lo (pMRVs)
http://www.pnas.org/content/early/2010/08/16/1006901107.full.pdf html

Urisman et al. (Including Silverman) (40% prostate cancer)
http://www.ncbi.nlm.nih.gov/pubmed/16609730?dopt=Abstract&holding=f1000,f1000m,isrctn

Switzer (detected XMRV in small number of prostate cancer patients in recent study)
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019065#abstract0

Singh (40% prostate cancer)
http://www.pnas.org/content/early/2009/09/04/0906922106.abstract
(Also in 25% of 178 breast cancer samples)
http://www.wipo.int/patentscope/search/en/WO2010132886

Arnold et al. (40% prostate cancer)
http://www.goldjournal.net/article/S0090-4295(10)00117-2/abstract

Danielson et al. (22% prostate cancer)
http://www.ncbi.nlm.nih.gov/pubmed/20936978

Fischer et al. (9.9% respiratory tract)
http://www.cdc.gov/eid/content/16/6/1000.htm

Maureen Hanson and Dr Bell (CFS)
Unpublished: Announced at the 1st International Workshop on XMRV
(XMRV found in 80% of severely ill patients, and 55% of all patients)
http://regist2.virology-education.com/abstractbook/2010_8.pdf

Kenny de Meirleir
Unpublished.
Blinded study, using the VIPdx to test for XMRV.
Patients tested were 57% positive for XMRV
http://www.investinme.org/IiME Conf...onal ME Conference 2011 Conference Report.htm

Wilfried Bieger (CFS) (Germany)
(Unpublished - announced at the IiME conference)
(40% of CFS patients)
http://www.investinme.org/IiME Conf...onal ME Conference 2011 Conference Report.htm

A Lithuanian group have published XMRV sequences in genbank.
http://forums.phoenixrising.me/showthread.php?11714-New-WPI-and-CDC-XMRV-sequences-in-genbank

Xenotropic Murine Leukemia Virusrelated Gammaretrovirus in Respiratory Tract
Nicole Fischer, Claudia Schulz, Kristin Stieler, Oliver Hohn, Christoph Lange, Christian Drosten, and Martin Aepfelbacher
June 2010
Emerg Infect Dis. 2010 Jun; DOI: 10.3201/eid1606.100066
http://www.cdc.gov/eid/content/16/6/1000.htm
(We detected XMRV in respiratory secretions of immunocompetent patients with and without RTI at a frequency of ?3.2%, which is in good concordance with the recently reported prevalence in the general population of up to 4%)

The Prevalence of XMRV in Healthy Blood Donors in Japan
XMRV reportedly found in 1.7% of random healthy blood donor samples from the blood supply in Japan, testing for antibodies only.
"Although our study had a limited sample size, the prevalence among blood donors as determined by identifying XMRV-specific antibodies was found to be 1.7%, while that among prostate cancer patients was found to be 6.3%"
(Publication status unknown to me)
http://www.diagnosesupport.com/heal...ted-virus-in&catid=132:xmrv-research&Itemid=8

A Preliminary Screening Of Xenotropic Murine Leukemia Virus-Related Virus In Japanese Prostate Cancer Patients
Tsukasa Igawa, Yoshinao Kubo, Katsura Kakoki, Naoki Yamamoto, Hideki Sakai
April 2011
The Journal of Urology (Abstract presented at the American Urological Association meeting)
http://www.jurology.com/article/S0022-5347(11)01208-0/fulltext
(MLV sequences were detected in four out of sixty one patients (6.7%))

Xenotropic Murine Leukemia Virus Related Virus (Xmrv) Is Present In Malignant Prostate Tissue But Does Not Affect Pathological Or Clinical Outcome
Chad Ritch, Ruslan Korets, Mireia Castillo-Martin, Josep Domingo-Domenech, Mitchell Benson, Carlos Cordon-Cardo
April 2011
The Journal of Urology (Abstract presented at the American Urological Association meeting)
http://www.jurology.com/article/S002...618-1/fulltext
Immunofluorescence: XMRV detected in 33/65 (51%) of malignant samples, and in only 5/42 benign samples (12%).
PCR: XMRV detected 14/65 (21%) of prostate cancer samples, and 2/42 (5%) in benign tissue
Combined PCR and IF: XMRV was detected in at least 17% of malignant samples and rarely found in benign tissue.


References

(1) No Association of Xenotropic Murine Leukemia Virus-Related Viruses with Prostate Cancer
Switzer.
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019065#abstract0
"Sequence analysis of the PCR-positive specimens was highly informative because it confirmed that all three specimens were XMRV-related. Also, the finding of a viral strain in three prostate cancer patients that is distinct from the XMRV seen in previous studies is significant and demonstrates a broader viral diversity. This would be an expected result consistent with virus evolution during spread and persistence."

(2) Human infection or lab artifact: will the real XMRV please stand up?
Silverman.
http://www.retrovirology.com/content/pdf/1742-4690-8-S1-A241.pdf

(3) XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors
Singh.
http://www.pnas.org/content/106/38/16351.full

(4) Interview with DR. ILA SINGH
"Not entirely sure, but there were different assays (e.g. immunohistochemistry) and different sample types (blood vs prostate tissue)."
http://www.cfscentral.com/2011/05/dr-ila-singh-we-are-now-convinced-that.html

(5) Mouse viruses and human disease
Gkikas Magiorkinis.
http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(11)70081-0/fulltext

(6) HTLV-1's replication: Perhaps it can explain XMRV's low degree of mutations
http://forums.phoenixrising.me/show...it-can-explain-XMRV-s-low-degree-of-mutations

(7)Blood Products Advisory Committee Meeting Transcript
Dr Harvey Alters Closing Remark at the Conference
http://www.investinme.org/InfoCentre Topics Dr Harvey Alter BPACM.htm

(8) Extensive Genetic Recombination in the XMRV Genome
Switzer.
http://retroconference.org/AbstractSearch/Default.aspx?Conf=20&Abs=40126

(9) Murine leukemia viruses (MuLV) and Xenotropic MuLV-related viruses exhibit inter-tropic complex recombination patterns
Mattia C F Prosperi , William M Switzer, Walid Heneine and Marco Salemi
6 June 2011
Unpublished abstract presented to the '15th International Conference on Human Retroviruses: HTLV and Related Viruses'
http://www.retrovirology.com/content/8/S1/A235

(10) Detection of MLV-related virus gene sequences in
blood of patients with chronic fatigue syndrome
and healthy blood donors

Alter & Lo.
http://www.pnas.org/content/early/2010/08/16/1006901107.full.pdf html

(11) Prevalence of XMRV in blood donors, HTLV and HIV cohorts
Abbott Diagnostics.
http://www.retrovirology.com/content/8/S1/A222

(12) Detection of MLV-like gag sequences in blood samples from a New York state CFS cohort
Hanson.
http://www.retrovirology.com/content/pdf/1742-4690-8-S1-A234.pdf

(13) Here are some of Harvey Alter's strong opinions regarding contamination.
This is not a very recent quote, but I haven't yet seen any indication that he has changed his opinions.
Blood Products Advisory Committee Meeting
Transcript
December 14, 2010
Dr Harvey Alters Closing Remark at the Conference
Invest in ME Website
http://www.investinme.org/InfoCentre Topics Dr Harvey Alter BPACM.htm
Extract:
"But I still want to counter by saying I think the current evidence for disease association is very strong, even though not universally confirmed. But it has been confirmed now in at least four studies, two of which were presented today, that either XMRV or a polytropic MLV is associated strongly with chronic fatigue syndrome. A point that I think was misrepresented today: In those labs who do find the agent, it is very reproducible. Judy has found the same patients to be positive by culture year after year. We have found a patient to come back after 15 years and still be positive. So this is not a single, isolated finding. It's confirmed by sequencing. It's reproducible over time."
(Please read the rest... It's interesting!)

(14) List of XMRV Research Studies
http://forums.phoenixrising.me/showthread.php?12518-List-of-XMRV-Research-Studies

(15) Unpublished XMRV studies and Future XMRV studies
http://phoenixrising.me/forums/showthread.php?12565-Unpublished-XMRV-studies-and-Future-XMRV-studies

(16) Recombinant Origin of the Retrovirus XMRV
Tobias Paprotka, Krista A. Delviks-Frankenberry, Oya Cingz, Anthony Martinez, Hsing-Jien Kung, Clifford G. Tepper, Wei-Shau Hu, Matthew J. Fivash Jr., John M. Coffin, Vinay K. Pathak
31 May 2011
Sciencexpress
http://www.sciencemag.org/content/early/2011/05/31/science.1205292.full.pdf

(17) XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors
Robert Schlaberga, Daniel J. Choeb, Kristy R. Browna, Harshwardhan M. Thakerb and Ila R. Singh,
September 8, 2009
PNAS
http://www.pnas.org/content/early/2009/09/04/0906922106.abstract

(18) New WPI and CDC XMRV sequences in genbank
http://forums.phoenixrising.me/showthread.php?11714-New-WPI-and-CDC-XMRV-sequences-in-genbank

(19) Addendum to the Science paper:
Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue Syndrome
http://www.landesbioscience.com/journals/virulence/MikovitisVIRU1-5.pdf

(20) Low Degree of Human T-Cell Leukemia/Lymphoma Virus Type I Genetic Drift In Vivo as a Means of Monitoring Viral Transmission and Movement of Ancient Human Populations
ANTOINE GESSAIN, ROBERT C. GALLO, AND GENOVEFFA FRANCHINI
21 October 1991
http://jvi.asm.org/cgi/reprint/66/4/2288

(21) Complete Nucleotide Sequence of a Highly Divergent Human T-Cell Leukemia (Lymphotropic) Virus Type I (HTLV-I) Variant from Melanesia: Genetic and Phylogenetic Relationship to HTLV-I Strains from Other Geographical Regions
ANTOINE GESSAIN, ENZO BOERI, RICHARD YANAGIHARA, ROBERT C. GALLO, AND GENOVEFFA FRANCHINI
12 November 1992
http://jvi.asm.org/cgi/reprint/67/2/1015.pdf

(22) The Low Evolutionary Rate of Human T-Cell Lymphotropic Virus Type-1 Confirmed by Analysis of Vertical Transmission Chains
S. Van Dooren, O. G. Pybus, M. Salemi, H.-F. Liu, P. Goubau, C. Remondegui, A. Talarmin, E. Gotuzzo, L. C. J. Alcantara, B. Galvo-Castro, and A.-M. Vandamme
January 22, 2004
Molecular Biology and Evolution
http://mbe.oxfordjournals.org/content/21/3/603.full

(23) A Comparison of Methods for the Detection and Association of XMRV in Chronic Fatigue Syndrome - Replication Analysis.
WPI
June 2011
http://files.me.com/jdj88/tzgfod
http://treatingxmrv.blogspot.com/2011/06/comparison-of-methods-for-detection-and.html

(24) Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.
Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, Malathi K, Magi-Galluzzi C, Tubbs RR, Ganem D, Silverman RH, DeRisi JL.
31st March 2006
http://www.ncbi.nlm.nih.gov/pubmed/16609730?dopt=Abstract&holding=f1000,f1000m,isrctn
(XMRV was first discovered in 2006 in prostate cancer patients. This is the original XMRV paper)
(Results: XMRV in QQ genotype = 40%, XMRV in RQ + RR genotypes = 1.5%, Healthy controls = not tested.)
  1. Bob
    Thanku for all of the positive feedback everyone. It makes it worthwhile doing it. :Retro smile:
  2. RustyJ
    Invaluable resource Bob. Intelligently and cogently written. Even I understood this. Easily the best summary on the subject I have read to date.

    i hope you can keep it up to date. Can I repost with attribution?
  3. fadingstar
    Thanks Bob....now if they could just stop fighting about whether it is or isn't and start figuring out what to do about it in the sick patients. sue
  4. currer
    Well done Bob for going through all this so systematically.
    Needless to say I agree with all of it!
  5. alex3619
    Hi Bob, the KDM data is likely to go higher for prevalence of XMRV, as he is doing biopsies now not just blood tests. Rumour, not substantiated to my satisfaction yet, has it that he is now finding it more often. Bye, Alex
  6. alex3619
    Bob, I waste a lot of time finding the same articles again and again, the result of my brain operating in the Twilight Zone. Having one article with lots of useful links will help a lot. Thank you. It should come as no surprise that I agree with your blog. Alex
  7. GaryK
    Bob your amazing! Brilliantly done. Thanks so much for all this effort.
    GaryK
  8. ukxmrv
    Bob, this is a magnificent effort.

    We owe you a beer, in fact a whole case of beers!

    I'm constantly looking for information or reminding myself which study said this.. and that.

    It's a real relief to find so much in one place and so well argued.

    Thank you!
  9. MEG
    Thank you so much. Amazing job.
  10. Enid
    Great to see this brought together Bob - many thanks for all your hard work. Can't comment as a non scientist but it seems to me XMRV just will not go away - the field and any conclusions are wide open still.