Conversing With Professor Myra McClure about XMRV and Contamination

Professor McClure

Professor Myra McClure is a retrovirologist from the Imperial College, London. Professor McClure exhibits an impressive resume encompassing a large portion of published studies: https://www1.imperial.ac.uk/medicine/people/m.mcclure/publications/

                                                                                                                                                                                                                                Professor McClure was a co-author of the paper published in Plos One in January 2010 titled, “Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome.” As the paper’s name suggests, this study found no evidence of XMRV or MLV in CFS patients or controls. This study can be found here: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0008519

                                                                                                                                                                                                                                Professor McClure has publically stated on many occasions that there is a high possibility that the XMRV/MLV related virus findings being implicated in CFS are a consequence of contamination.

 

The Usefulness of Critiquing Professor McClure’s Statements

It can be argued that Professor McClure is just an arbitrary retrovirologist whose opinions on XMRV/MLV related viruses will not have an influence on the final scientific consensus. This is true and Professor McClure has publically stated that “Nothing on God’s Earth could persuade me to do more research on CFS.” The XMRV/MLV related virus- CFS paradigm shift will likely stem from several USA government departments working in unison. The question is then posed, “What is the value is criticising Professor McClure’s viewpoint?”

                                                                                                                                                                                                                                   Professor McClure has recently been the leading proselyte reviving the contamination hypothesis as an explanation for the Lombardi et al. results. She has appeared in a flurry of media interviews and seems to be driven by the purpose of spreading the contamination hypothesis. She has recently appeared in a Medpage Today interview, addressed the American Society for Microbiology, been interview for This Week in Virology and presented the contamination argument at the International XMRV conference. Professor McClure’s recent string of media interviews and conference talks will unquestionably influence the short-term research plans of some scientists. She has made ad nausium contamination accusations in her talks although some of these accusations are subtle and contamination is implied. Professor McClure’s underwhelming ‘Science Media Centre’ press release synopsis of the Lo et al. study may have contributed to the UK media boycott of the story that was covered by hundreds of media groups worldwide (this press release can be found here: http://www.sciencemediacentre.org/pages/press_releases/10-08-23_cfs_virus_pnas.htm)  Professor McClure has undeniably had a significant number of scientists, ME/CFS patients and the public hear her comments.

 

Professor McClure’s contamination accusations would be useful if there was a high likelihood of contamination in this situation. If contamination was the cause of the XMRV/MLV results then her media interviews may be of benefit due to

1. Reducing the number of ME/CFS patients taking anti-retrovirals
2. Inhibiting the false hope that the ME/CFS community has somewhat projected on the XMRV/MLV outcome.
3. Stopping research scientists waste their funds on research that is based on contamination

 

If the contamination likelihood was negligible, then Professor McClure’s recent media comments may have caused some harm. If contamination was not the cause of the XMRV/MLV results then her media interviews and conferences may be counterproductive due to:

1. Causing scientists to delay XMRV/MLV research studies
2. Causing patients to delay taking anti-retrovirals when this may be a useful treatment (although this is also contingent on XMRV/MLV causing CFS which is a separate debate.)
3. Causing scientist such as Dr. Mikovits to focus her attention on disproving the contamination theory rather than focusing on the pathogenesis and possible treatment of XMRV/MLV
4. Delaying the paradigm shifting realisation that ME/CFS is a purely organic illness.
5. Short term research projects reconfirming the Lo et al. and Lombardi et al. results rather than researching pathogenesis and possible treatment of XMRV/MLV
6. Dashing the hopes of ME/CFS XMRV/MLV positive patients by ruling out this as the etiology of their disease.
7. Focusing the debate on the presence of XMRV/MLV in ME/CFS patients. This is as opposed to the more pragmatic debate concerning whether XMRV/MLV causes or plays some role in the development of ME/CFS.

                                                                                                                                                                                                                                                 I have presented a somewhat simplified analysis above. The actual implications of Professor McClures’ talks may be more subtle and nuanced. Essentially the usefulness of Professor McClure’s remarks are contingent on whether the XMRV/MLV results are due to contamination. As the link between XMRV/MLV and ME/CFS has not been ruled out and contamination has not been ruled out definitively then the usefulness of Professor McClure’s comments can be assessed according to the likelihood of contamination causing the aforementioned results. I will attempt to determine the likelihood of this contamination hypothesis being correct and consequently whether Professor McClure’s comments are justified and productive.

 

My First Email to Professor McClure

On the 15^th of September 2010 I emailed Professor McClure, posing six questions.

1.     XMRV from patient samples has been seen from a transmission electron microscope. Does this rule out contamination?

2.    XMRV has been grown in a petri dish and uninfected human cells have been infected by it. Does this rule out contamination?

3.    The Lo et al. paper saw the MLV related virus mutate over a 15 year period in 7 individuals. Does this rule out contamination?

4.    XMRV has produced antibodies in CFS patients. Does this rule out contamination?

5.    XMRV from ME/CFS patient samples has been sequenced and it has been deemed to be distinct from every known murine ERV. It is almost identical to XMRV found in prostate cancer patients. Does this rule out contamination?

6.    You seem open to the possibility of XMRV/MLV related virus and the ME/CFS association being a product of contamination but not the XMRV-prostate cancer connection being due to contamination. What is the reason for this?

 

Professor McClure responds to my First Email

On the 16^th of September 2010, Professor McClure responded to my questions. I’d like to thank Professor McClure for taking time to reply to my emails. Professor McClure’s response appears below.

“The answer to all the questions you pose , with the exception of the last is, No. In answer to your last question, the flanking sequeces to the integration sites for XMRV in prostate cancer cells have been shown to be human sequeces and not murine, suggesting this is not a laboratory contaminant.”

 

I was perhaps rather naive in my definitive phrasing of the questions that I posed to Professor McClure. In hindsight I would have phrased the question aspect differently.

 

My Second Email to Professor McClure

I posed the question pertaining to the accumulation of evidence of the following five factors:

1.      “XMRV from patient samples has been seen from a transmission electron microscope.

2.         XMRV has been grown in a petri dish and uninfected human cells have been infected by it.

3.    The Alter et al. paper saw the MLV related virus mutate over a 15 year period in 7 individuals.

4.    XMRV has produced antibodies in CFS patients.

5.    XMRV from ME/CFS patient samples has been sequenced and it has been deemed to be distinct from every known murine ERV. It is almost identical to XMRV found in prostate cancer patients”.

I then wrote,

“Do you believe that

• the totality in weight of the aforementioned 5 factors
• in combination with the non-ubiquitous and comparable levels of MLV related virus in CFS patients and controls from several studies (and almost a dozen independent research institutions)
• also in combination with the multiple negative contamination tests including Switzer’s real time PCR assay (performed on the WPI samples) with two fluorescent mouse mitochondria DNA specific probes

provide solid evidence against the contamination possibility? If no, then why not?”

 

Professor McClure’s Response to my Second Email

 “I wish I had time to go into detail, but to answer your question directly. I believe the weight of evidence indicates laboratory contamination. There have now been 4 major papers published in reputable journals from highly reputable laboratories who have been unable to confirm the Lombardi findings. In fact, if you read the Bill Switzer paper from CDC, you will see that this paper actually describes, not just one study from CDC, but 3 since they had the samples independently tested in 3 laboratories (and blinded the samples). This effectively means that there were 3 studies described in this paper, all giving the same negative result . Hence, although I say 4 papers, the total number of investigations is actually 6.  I would think that the onus is now on the Whittemore Institute to prove beyond all doubt that they are right when the rest of the world cannot reproduce their findings.

 

My view (confirmed at the XMRV workshop) is that the US government agencies (NIH, CDC, FDA) will organise multi-centre trials with a blinded and randomised panel of samples and reagents in the near future and this will settle the matter once and for all.

 

I do believe that part of the problem for the misleading results in both the Science paper and the PNAS paper by Lo et al is the fact that CFS samples and control samples have not been blinded, randomised and treated in exactly the same way at the same time. Lo et al say their samples were “coded”, but that might just mean that the patient identity was removed, it doesn’t say that the samples and controls were simultaneously subjected to the same  procedures and that the person doing the experiments was blinded as to which were CFS samples and which were controls. In fact, if you look at Fig 1 and Fig 2 in this paper, you can see that a different primer set was used on the CFS patient samples and the control samples. This suggests that the study was not blinded. 

 

Professor Robin Weiss who is the UK’s leading retrovirologist has a very readable article coming out in the next couple of weeks addressing why different laboratories are getting different results and discussing the problems  retrovirologists face in isolating contaminating viruses that they think are new human viruses. I have copied my PA into this reply, so that when the article appears in print, she can send a copy to you. If nothing else, it will give you information about how claims like this are not a new phenomenon in retrovirology and the steps that were taken previously to settle similar arguments. I think you will find it interesting.”

 

My Analysis of Professor McClure’s Arguments

Let me preface this section by stating that I am not very well versed in retrovirology and I apologise in advance if I have made any erroneous statements pertaining to the retrovirology specifics.

 

“I believe the weight of evidence indicates laboratory contamination.”

During the most recent ‘This week in Virology’ podcast, Professor McClure made several comments that reiterate her above opinion. These include,

“There is no point in me going back (to the laboratory) I’d just get the same result.”

“I have no plans to revisit those samples because I’m quite confident of the result and the result we would get.”

“Nevertheless in the interests of science, I’ve asked if we could get a few fresh blood samples from chronic fatigue (sic) patients. Following all of the recommendations which are now coming out from the Whittemore Peterson (sic) and we are getting sort of drip fed little hints of how to do it, then I’m very happy to try and I’m very happy to say alright by this method we can find it if we can find it but you know, I have err, I have doubts about that.”

                                                                                                                                                                                                                                Professor McClure seems to support the hypothesis that both the Lo et al. study and the Lombardi et al. study found a CFS link to XMRV/MLV due to contamination. I will now examine “the weight of evidence” of the Lo et al. study and determine if the study processes and results “indicate laboratory contamination.”

                                                                                                                                                                                                                                             The Lo et al. study provides several arguments against the contamination claims. These include specific tests and steps that Lo et al. performed in order to rule out contamination.

1. Every sample in the Lo et al. study that tested MLV positive (based on gag gene sequences), was tested for traces of mouse DNA contamination. The PCR used in this MLV contamination test was designed so that it was more sensitive to registering mouse DNA than MLV gag sequences. It follow that if MLV by contamination was detected, mouse DNA would have also been detected. This PCR contamination test didn’t detect any mouse DNA in the reaction mixtures or clinical samples included in the study. Lo et al. concluded that this rules out the possibility of contamination by mouse DNA.
2. Lo et al. then assessed the likelihood that the clinical samples or assays used may have been contaminated. The blood samples used in this study were obtained from clinical laboratories that had never contained or worked with mice or retroviral vectors. The blood from these clinical samples was drawn from needles into empty vacuum tubes that were only opened for the blood drawing process. The Lo et al. laboratory in which the PCR tests were performed had also never worked with murine cells, murine blood samples or MLV vectors.
3. The MLV gag sequences that were detected in the study cohort’s blood comprised at least six distinct types of MLV sequence. Lo et al. hypothesise that if contamination is the mechanism behind results then the same sequences arise in most, if not all of the positive samples. The Lo et al. study also determined that these six plus sequences had “significant variations” when compared to all other known exogenous MLVs and viral vectors.
4. This Lo et al. study also set up 300 negative controls for the multiple PCR amplification aspect of the study. This step involved attempting to detect the MLV gag gene however all results were negative.
5. Lo et al. also strengthened their anti-contamination argument by obtaining fresh blood samples from 8 patients. These fresh samples were obtained approximately 15 years after the original drawing of the samples. Seven out of the eight patients still had MLV gag sequences detected. Lo et al. found that these newly obtained sequences were significantly varied from the original patient sequences. Lo et al. wrote that this “would be expected in retroviral infections, but not from contamination.”

 

The two lead study authors, Dr. Lo and Dr. Alter have publically made several statements emphasising their confidence in the result they obtained. Dr Alter made one of these comments to CFS Central, “There were no changes in the conclusions, but we added data that made the conclusions stronger…For one thing, we did some further work to feel confident that there was no contamination.” (Dr Alter is referring to the delay in publication of the Lo et al. study.) (http://www.cfscentral.com/2010/08/fdanihharvard-xmrv-study-same-thing.html)

 

During the study’s teleconference, Dr. Alter said, “The retroviruses…exist in big families of viruses…Hepatitis C is a very good example, nobody’s infected with one variant… they have a whole huge family of variants…and if you take any given patient the patient will have multiple variants in them, and different patients will have different variants, so it’s very characteristic of these kind of RNA viruses.” These comments emphasise the MLV sequence heterogeneity, as found by Lo et al., that is typical of a retroviral infection and not of contamination.
Lombardi et al. went to equally as impressive measure as Lo et al. to discredit the contamination theory explaining their own studies results. I can no longer access the Lombardi et al. paper in its entirety hence I cannot explain in detail the contamination- specific tests that Lombardi et al. performed. Despite this, I can state that all of the Lombardi et al. tests came back negative for contamination. Dr. Switzer, lead author of the CDC’s study that failed to detect XMRV/MLV designed his own contamination test that was performed on the Lombardi et al. samples. This test was a real time PCR assay that contained two fluorescent mouse mitochondria DNA specific probes. This test, created by Dr. Switzer failed to detect any signs of contamination amongst the Lombardi et al. samples.

 

There is no evidence that the Lombardi et al and Lo et al. studies were contaminated and all of the subtests in these studies came up negative for contamination. Both studies took every precaution to avoid contamination and to test for contamination. To be justified in believing that “the weight of evidence indicates laboratory contamination” one would expect at least one positive test result for contamination in the aforementioned tests. In a subsequent section I will also address Professor McClure’s statement that “The weight of evidence indicates laboratory contamination.” This subsequent section is titled, ‘Explanatory Power and Mechanisms.’

 

“There have now been 4 major papers published in reputable journals from highly reputable laboratories who have been unable to confirm the Lombardi findings. In fact, if you read the Bill Switzer paper from CDC, you will see that this paper actually describes, not just one study from CDC, but 3 since they had the samples independently tested in 3 laboratories (and blinded the samples). This effectively means that there were 3 studies described in this paper, all giving the same negative result . Hence, although I say 4 papers, the total number of investigations is actually 6.”  Professor McClure recently gave a Medpage Today interview (found here: http://www.medpagetoday.com/MeetingCoverage/ICAAC/22166) During this interview she stated that “there’s no point in having a league table of those who can find the virus and those who can’t find the virus and see who wins out.” Although Professor McClure doesn’t implicitly state this in the above bolded quote, she does imply that the negative studies are ahead, according to her own “league table.” The very fact that Professor McClure mentions the several premises in the bolded quote implies the conclusion that there are many more studies not finding XMRV/MLV than there are finding XMRV/MLV. Professor McClure may argue that this implied conclusion was not her intention hence I will examine her quote.

                                                                                                                                                                                                                                   Professor McClure’s bolded quote is a series of mainly factual statements (some are normative, including the sentences containing “reputable journals” and “highly reputable laboratories.”) If no conclusion is implied from her statements, then they are superfluous statements that she has added for no purpose. Professor McClure may argue that scientific statements are factual hence her quote. This is true however scientific ‘factual’ statements require the author to draw conclusions from the statements. This is part of the reason why scientific journal articles include a section for ‘discussion’ and a separate section for ‘conclusion.’ Scientific and factual statements that are used in an argument should be tied in to the author’s conclusion. If they are arbitrarily added to an argument and not linked to a sub-conclusion or a conclusion then they can be deemed as superfluous and irrelevant.

                                                                                                                                                                                                                                            Her statement “Hence, although I say 4 papers, the total number of investigations is actually 6” may be deemed to be the sub-conclusion of her previous premises. This ‘sub-conclusion’ does not relate to the rest of her argument unless the implied conclusion that the negative papers have more weight than the positive papers is introduced. Professor McClure’s other statement which may be a sub-conclusion is “There have now been 4 major papers published.” She does not relate this to her argument that contamination is a likely hypothesis.

                                                                                                                                                                                                                                Professor McClure’s above full bolded quote is therefore either

1. Superfluous and irrelevant as she does not link it with her conclusion, or
2. Has an implied conclusion that the negative XMRV studies are leading the positive studies and this supports the contamination hypothesis.

                                                                                                                                                                                                                                                If 1. is the case then there is no purpose in my analysis of her quote due to its non-relation to her overall conclusion. If 2. is the case then she has committed an explicit contradiction. This is due to her quote in the Medpage interview that “There’s no point in having a league table of those who can find the virus and those who can’t find the virus and see who wins out.”

                                                                                                                                                                                                                                        Despite this conclusion I have reached pertaining to Professor McClure’s quote, I will now analyse it from an accuracy perspective. “There have now been 4 major papers published in reputable journals from highly reputable laboratories who have been unable to confirm the Lombardi findings.” Professor McClure has used the terms “reputable journals” and “highly reputable laboratories” in a meliorative manner to support the implied conclusion that studies performed by quality laboratories, that are peer reviewed and accepted by eminent journals would have accurate results. This is not an argument for accepting a scientific paradigm. The studies should be judged on science alone, regardless of the highbrow nature of the institution or journal. Having said this, there is a general correlation between accuracy of science articles and the impact of the journal they are published in. I will therefore examine the impact factors of the XMRV related studies. The Lombardi et al. study and the Lo et al. study were published in journals with a higher impact factor than the negative studies. The top 5 scientific journals in descending order of Impact factor are 1. Nature (51.97), 2. Science (48.78), 3. New England Journal of Medicine (19.84), Cell (15.34), PNAS (14.88). The Lombardi et al. study was published in Science, the second highest impact rating journal and the Lo et al. study was published in PNAS, the fourth highest impact rating journal. None of the XMRV negative studies were published in the top 5 journals. The CDC study that Professor McClure perpetually mentions in her quote was published in ‘Retrovirology’ which has an impact factor of 4. I am not claiming that the journals the studies were published in necessarily influences their validity. I am only countering Professor McClure’s implied argument that the “reputable” nature of the journals of the negative XMRV studies adds to their validity over the positive XMRV/MLV studies.

                                                                                                                                                                                                                                Professor McClure alludes to the “highly reputable laboratories” that failed to detect XMRV/MLV. Similar to the aforementioned journal argument, the quality of laboratories (if one can measure or distinguish reputable laboratories) does not automatically add to the validity of the findings. Professor McClure’s quote seems to subtly imply that the quality of the laboratories not detecting XMRV/MLV is higher than the quality of the laboratories that can detect XMRV/MLV. She also fails to mention the laboratories other than the Whittemore Peterson Institute (WPI) that detected XMRV/MLV. The positive Lombardi et al. study was performed by the not only the WPI but also the National Cancer Institute and the Cleveland Clinic. The positive Lo et al. study was performed by both the National Institute of Health and the U.S. Food and Drug Administration. These laboratories are also considered to be “highly reputable” by many people within the scientific domain.

                                                                                                                                                                                                                                              “In fact, if you read the Bill Switzer paper from CDC, you will see that this paper actually describes, not just one study from CDC, but 3 since they had the samples independently tested in 3 laboratories (and blinded the samples). This effectively means that there were 3 studies described in this paper, all giving the same negative result .” This statement by Professor McClure uses the logic that three independent laboratories testing samples in their own laboratories under the one study umbrella can be counted as three separate studies. Using Professor McClure’s own logic, the Lombardi et al. study has been confirmed twice. The WPI, NCI and CC all performed unique tests forming the Lombardi et al. study at their own laboratories. Professor McClure’s own logic has therefore deemed that the Lombardi et al. study has been confirmed twice. During a recent American Society of Microbiology meeting, Professor McClure has stated that “So my position would be that in the absence of confirmatory evidence, there is… no confirmatory link of CFS and either of these Murine Leukemia Viruses. But of course, life changes and if there is evidence to the contrary then of course we would happily embrace it.” Using Professor McClure’s own logic that three laboratories performing tests under the one study umbrella counts as three separate studies, then the Lombardi et al. study has been confirmed twice. This confirmation is what Professor McClure stated she required to convince her of the XMRV-CFS connection. Therefore Professor McClure should believe the XMRV-CFS connection.

                                                                                                                                                                                                                                   Professor McClure made the statement that “There have now been 4 major papers published in reputable journals from highly reputable laboratories who have been unable to confirm the Lombardi findings ….Hence, although I say 4 papers, the total number of investigations is actually 6.” This statement seems to appeal (through the means of implying) to the high number of negative XMRV studies as evidence for the contamination hypothesis. Professor McClure has used the fallacy, argumentum ad numerum. Although multiple similar studies generally provide more weight for a specific outcome, multiple studies shouldn’t be cited as definitive proof of an outcome. This is especially true of studies which fail to detect anything, such as the negative XMRV studies. Due to the current discrepancy between the XMRV negative and XMRV positive results, a scientific consensus will likely be reached when a verifiable mechanism has been determined providing an explanation for the current discrepancy in XMRV results.

                                                                                                                                                                                                                                Professor McClure’s above quote is also problematic due to the precise nature of the negative XMRV studies. These negative studies have used dubious cohorts, flawed methodologies and questionable blood collection methods. I have detailed these erroneous aspects of the negative XMRV studies in detail elsewhere on this blog hence I will not cover these issues again here. If a flawed methodology is used ad nauseum, it does not add weight to the validity of the result. Albert Einstein made two quotes supporting the notion that I have mentioned here. Einstein said “Insanity is doing the same thing over and over again and expecting different results.” Also when a pamphlet was published titled ‘100 authors against Einstein,’ he retorted “If I were wrong, one would be enough.” These quotes emphasise the danger of using the argumentum ad numerum fallacy that Professor McClure has implied by her quote. 

 

“I would think that the onus is now on the Whittemore Institute to prove beyond all doubt that they are right when the rest of the world cannot reproduce their findings.”

Professor McClure was recently interview by ABC radio in Australia. The following is a transcript of part of her interview:

“Norman Swan: And it’s not possible it’s a contaminant that it’s just crept into samples by accident? ( Norman Swan is referring to prostate cancer.)

Myra McClure: That’s always a problem with retroviruses and their association with disease – this time it’s not and I’ll tell you why, because the people who published the American studies have been very careful to sequence this virus and the sequence and the phylogenetic analysis that is looking at the sequence of one virus compared to another and the eight samples they have are sequences taken from eight different viruses, taken from eight different patients with prostate cancer. All these are slightly different. Now if it were a lab contaminant you would have expected them all to be the same and they’re not.

And secondly if it were a lab contaminant it would be exactly the same in sequence as all other known murine leukaemia viruses and it’s not – it’s different. So it’s a real new virus.”

This interview can be found here: http://www.abc.net.au/rn/healthreport/stories/2010/2867629.htm#transcript

                                                                                                                                                                                                                                               In the above argument, Professor McClure has proposed that two factors are required for retroviral association to be distinguished from contamination. She states that these two factors are:

1. The varying retroviral sequences among the set of Z-retrovirus positive patients.
2. The retroviral sequences distinctiveness when compared to the set of known contaminants (in this case, murine leukemia viruses.)

According to Professor McClure’s argument, these are the two necessary conditions that need fulfilling. If these criteria are fulfilled then “It’s not possible it’s a contaminant.”

                                                                                                                                                                                                                                               In the above argument, Professor McClure is arguing against the prostate cancer XMRV association arising due to contamination. If her argument concerning ruling out contamination is superimposed onto the CFS- XMRV association then she is ruling out an XMRV- CFS contamination. This is due to the XMRV discovered in CFS patients fulfilling both of the criteria that Professor McClure herself has established to rule out contamination. The XMRV found in CFS patients is 1. Different among the CFS patients, determined by sequencing and 2. Is distinct from all known murine leukemia viruses.

                                                                                                                                                                                                                                    Professor McClure now has two options to remain in a consistent position.

1. She changes her position to state that the XMRV found in prostate cancer may be due to contamination.
2. She changes her position to rule out the XMRV contamination hypothesis in CFS.

                                                                                                                                                                                                                                                   I can anticipate that Professor McClure would not change her position on these two illness associations with XMRV but rather use a post hoc fallacy. This would entail claiming that more criteria are necessary for contamination to be ruled out such as showing that the retrovirus detected contains human not murine sequences. For Professor McClure to establish this third criterion she would be modifying her original objective stipulations in order to retain a consistent position for an evidence based stance. This is the antithesis of science, which works by evidence based stances becoming theories due to fitting in with already established criteria.

                                                                                                                                                                                                                                     Professor McClure has presented an argument pertaining to necessary conditions to rule out contamination and this argument supports the notion that the CFS-XMRV association has not arisen due to contamination. In essence, Professor McClure’s own argument provides further support for the XMRV association to CFS not being due to contamination.

 

“I would think that the onus is now on the Whittemore Institute (sic) to prove beyond all doubt that they are right when the rest of the world cannot reproduce their findings.”

                                                                                                                                                                                                                                  Professor McClure’s above quote refers to the onus (or burden) of proof as now being on the WPI. Burden of proof challenges and debates concerning who is required to prove the burden of proof are common in academia. These debates often involve a back and forth argument in order to determine which side the burden of proof falls. In many instances no consensus is reached on who holds the burden of proof. Often these burden of proof arguments are laborious and spurious. The burden of proof in most instances falls on the group making the most outrageous claim. In the XMRV-contamination context, determining which group is making the more outrageous claim is not a simple matter. One can argue that by linking XMRV and CFS, one is proposing a paradigm shift which would require a burden of proof. It can be counter argued that many laboratories have sufficiently tested the blood samples for contamination and all tests have been negative. Consequently the burden of proof falls on those contamination proponents to provide a test to detect contamination. The burden of proof argument in this situation is not as black and white as Professor McClure’s claim that “the onus is now on the Whittemore Institute (sic) to prove beyond all doubt that they are right.”

                                                                                                                                                                                                                                                A burden of proof argument isn’t entirely necessary in this situation as the US government’s blood working group are currently working to determine why discrepancies are occurring between different scientist’s XMRV results. By referring to the WPI’s burden of proof, it can be argued that Professor McClure is setting up a spurious argument that will inevitably end in deadlock.

                                                                                                                                                                                                                                    Professor McClure’s statement that “the onus is now on the Whittemore Institute (sic)” fails to accurately provide an assessment of the situation. The NIH, FDA, CC and NCI have all detected XMRV/MLV in CFS patients in high percentages. By erroneously singling out the WPI, Professor McClure presents what is a pseudo-enhanced argument. If her statement accurately referred to the totality of the five separate laboratories, the reader may not be as swayed by her argument.

                                                                                                                                                                                                                                    Professor McClure wrote that “The rest of the world cannot reproduce their (the WPI’s) findings.” She uses the phrase “rest of the world” hyperbolically. This phrase is an erroneous way to describe a handful of laboratories. The reader may be influenced by Professor McClure’s use of this dysphemism. It sounds impressive to state “rest of the world” however in this context, this is a synonym for “several laboratories.” It is also erroneous to claim that other laboratories cannot reproduce the Lombardi et al. study and I expand on this point under the subsequent heading titled ‘Confirmation.’

                                                                                                                                                                                                                                    Scientific journals are often an accurate gauge relating to the burden of proof. The Science and PNAS journals both instructed the XMRV/MLV positive laboratories to perform additional tests to rule out contamination. Both Lo et al. and Lombardi et al. performed these additional tests which came back negative for contamination. Science and PNAS both accepted the high XMRV/MLV prevalence in CFS patients study results. It can be argued that this acceptance and consequential publication, a process that lasted many months, was the defining factor that removed the burden of proof from the XMRV/MLV positive study authors. Both journals’ actions of publishing the XMRV/MLV studies indicate that they believe the contamination possibility has been appropriately addressed.

                                                                                                                                                                                                                                                It can be argued that the study authors’ that have found an XMRV/MLV-CFS correlation have performed every possible test to exclude contamination hence how else can they prove the burden of proof? In her first response to my emails, Professor McClure described the reason why she doubted that the prostate cancer-XMRV/MLV connection was spurious. She wrote, “The flanking sequeces to the integration sites for XMRV in prostate cancer cells have been shown to be human sequeces and not murine, suggesting this is not a laboratory contaminant.” The question then arises as to why the CFS- XMRV/MLV positive study authors’ haven’t performed this step on the CFS patient samples. It is not through complacency or avoiding the issue that these scientists have not to date performed this test. It is a very laborious process to identify provirus integration sites. Lo et al. have estimated that there is only a single virus gene copy per 400-4000 nucleated peripheral blood mononuclear cells. Lo et al. highlight a second problem with identifying the provirus integration sites- Previous studies have found that these sites vary significantly. It has been speculated that some scientists are currently working on the laborious process of demonstrating integration of MLV genes into the human genome of CFS patients.

                                                                                                                                                                                                                      Confirmation

During a recent American Society of Microbiology meeting, Professor McClure made several remarks alluding to the Lo et al. study not being confirmatory of the Lombardi et al. study. She said, “It’s important to note that this second paper coming out of the FDA cannot be used at confirmatory evidence of the initial paper that was published in Science and we just have to wait and see whether this paper is confirmed by others.” During the latest ‘This Week in Virology’ podcast, Professor McClure reiterated these sentiments, “It’s important to point out that they (Lo et al.) have not found the same virus either. They have found another member of the family of endogenous retroviruses. Theirs is a polytropic as opposed to a xenotropic virus.”

                                                                                                                                                                                                                                Professor McClure’s comments that the Lo et al. paper was not confirmatory of the Lombardi et al. paper are disputed by the two lead authors of the Lo et al. paper- Dr. Lo and Dr. Alter. Dr Alter has stated that “We think, basically, it confirms the findings of the Whittemore-Peterson Group.” Dr Alter continues on to state that “It does, at least, confirm the findings of Whittemore-Peterson Institute…I think one wants to go back to their studies because they’ve had more time, and they’ve done extensive work…so their study is more advanced than ours, but -with that as…er…with them having done the groundwork I think our study is highly confirmatory of their work…” These comments by Dr Alter emphasise the Lo et al. study authors’ thoughts that their study confirms the Lombardi et al. study.

                                                                                                                                                                                                                                   Professor McClure may argue that Lo et al. detected a different type of Murine Leukemia Virus related virus compared to Lombardi et al. hence the study is not confirmatory. Dr. Lo emphasises that detection of a variety of MLV sequences would be expected in a retroviral infection. Dr. Lo states, “that’s exactly what we anticipate for retrovirus infection…over time you will see the many different sequences there.” Dr. Lo also states that “We can say we found this kind of variability that’s actually more consistent with the natural form of a retrovirus infection in this group of patients.” Dr. Lo’s comments reiterate the point that the diversity of MLV’s being detected in CFS patients makes a stronger case for retroviral involvement in the illness, not a weaker case.

                                                                                                                                                                                                                                               Dr. Lo has also commented that “They are in the same family but…they are compatible with the earlier finding of the XMRV, however they are not identical and they are more diverse.” This quote highlights the Lo et al. studies extension of the Lombardi et al. study through the means of detecting a variety of MLV in CFS patients. Dr. Alter clarifies that the WPI have also detected a range of MLV in CFS patients since the publication of the Lombardi et al. study, “Since the original publication, the WPI and NCI groups have found that they too are finding greater variability in their patients so it is not just XMRV as in the original cohort of patients.” As both the Lo et al. group and the Lombardi et al. group are finding a range of MLV’s in CFS patients and both study’s authors believe that the Lo et al. group have confirmed the Lombardi et al. result, Professor McClure’s statements are quite contentious.

                                                                                                                                                                                                                                          During the recent American Society of Microbiology meeting, Professor McClure stated “So my position would be that in the absence of confirmatory evidence, there is at the minute no confirmatory link of CFS and either of these Murine Leukemia Viruses. But of course, life changes and if there is evidence to the contrary then of course we would happily embrace it.” This quote by Professor McClure seems to indicate that if a positive MLV-CFS study is confirmed, then she will believe in the CFS-MLV link. As I have documented above, the Lo et al. study can be considered as a confirmation study. Also the three autonomous laboratories that produced the Lombardi et al. paper can be considered as two confirmations of the first laboratories results. Also the Lo et al. paper was performed by two laboratories hence it can be argued that the second laboratory confirmed the first laboratories result. This is due to Professor McClure’s logic that “In fact, if you read the Bill Switzer paper from CDC, you will see that this paper actually describes, not just one study from CDC, but 3 since they had the samples independently tested in 3 laboratories (and blinded the samples). This effectively means that there were 3 studies described in this paper, all giving the same negative result . Hence, although I say 4 papers, the total number of investigations is actually 6.”

                                                                                                                                                                                                                                                If one ignores the five separate laboratories finding MLV in a significant number of CFS patients and the Lo et al. study being confirmatory of the Lombardi et al. study, another confirmation study has emerged. This study was revealed at the recent XMRV International Conference. This study by Hanson et al. was performed at Cornell University and concluded that “Our results corroborate those of Lombardi et al.” This Hanson et al. study also went to great lengths to avoid contamination. The study abstract states that “PCR with mouse COX2 primers was performed on all cDNA preparations to rule out mouse cell contamination.” The laboratory in which this study was performed had never worked with or housed mice.

                                                                                                                                                                                                                                              Let us assume that Professor McClure is given the benefit of the doubt and contrary to the study’s authors, the Lo et al. study is not considered confirmatory of the Lombardi et al. study. Also if Professor McClure’s logic concerning umbrella studies counting as several studies is only applied to the Switzer et al. study and not the Lombardi et al. or Lo et al. studies then the Hanson et al. study is still confirmatory of the Lombardi et al. study. Professor McClure’s has made an admission that “In the absence of confirmatory evidence, there is at the minute no confirmatory link of CFS and either of these Murine Leukemia Viruses. But of course, life changes and if there is evidence to the contrary then of course we would happily embrace it.” Professor McClure should therefore “happily embrace” the CFS- XMRV/MLV connection.

                                                                                                                                                                                                                                Professor McClure made the comment in her response to my second email that “I would think that the onus is now on the Whittemore Institute (sic) to prove beyond all doubt that they are right when the rest of the world cannot reproduce their findings.” She seems content to segregate the WPI from the other laboratories finding XMRV as her quote indicates that no portion of the Lombardi et al. study has been reproduced. In her email to me, Professor McClure also made comments indicating a definitive line creating a dichotomy between the studies finding no XMRV in controls or CFS patients and the studies finding XMRV in CFS patients, “There have now been 4 major papers published in reputable journals from highly reputable laboratories who have been unable to confirm the Lombardi findings…. I would think that the onus is now on the Whittemore Institute to prove beyond all doubt that they are right when the rest of the world cannot reproduce their findings.” Professor McClure has committed the fallacy of creating a false-dichotomy. This fallacy is defined as “A situation in which two alternative points of views are presented as the only options, whereas others are available.” Professor McClure’s false-dichotomy has excluded those studies which have found a comparable percentage of XMRV in healthy controls or controls to the Lombardi et al. study. These studies did not test for XMRV in CFS patients however they are in contradiction to the studies finding no XMRV in CFS patients or controls.

 

The first of these studies is a German study (Fisher et al.) which detected XMRV in respiratory secretions. XMRV was detected in 2.3% of people with respiratory infections. Also XMRV was detected in 3.2% of people with chronic obstructive pulmonary disease. Japanese scientists detected XMRV in 1.8% of healthy blood donor’s blood. XMRV was also found by Chinese scientists at a prevalence of 3.4% in Chinese blood donors. These percentages are comparable to Lombardi et al. group which detected XMRV in 3.8% of healthy controls. The single figure detection of XMRV in controls of several studies is a partial confirmation of the Lombardi et al. study. This single figure positive percentage confirms an aspect of the Lombardi et al. study. Professor McClure may argue that there is still a range of percentages being found in these studies hence it is erroneous to deem this aspect of the study as confirmatory. Retroviruses are not distributed homogenously worldwide and retrovirus prevalence fluctuates depending on the area. Also a non-standardised XMRV test may cause a small discrepancy in results due to some false negatives being detected. A third factor explaining the small discrepancy in results is the small number of controls being used on some of the studies which may influence the actual XMRV positive percentage of a larger group or country. When Professor McClure portrays the situation as the WPI versus “the rest of the world” she is creating a false-dichotomy and as I have demonstrated the situation resembles more of a nuanced trichotomy than the dichotomy Professor McClure suggests.

 

 

“I do believe that part of the problem for the misleading results in both the Science paper and the PNAS paper by Lo et al is the fact that CFS samples and control samples have not been blinded, randomised and treated in exactly the same way at the same time. Lo et al say their samples were “coded”, but that might just mean that the patient identity was removed, it doesn’t say that the samples and controls were simultaneously subjected to the same  procedures and that the person doing the experiments was blinded as to which were CFS samples and which were controls. In fact, if you look at Fig 1 and Fig 2 in this paper, you can see that a different primer set was used on the CFS patient samples and the control samples. This suggests that the study was not blinded.”  

                                                                                                                                                                                                                                Professor McClure has stated that the Science and PNAS papers had “CFS samples and control samples (that) have not been blinded, randomised and treated in exactly the same way at the same time.” This argument is used by Professor McClure as a potential mechanism to explain how the CFS samples were contaminated while the control samples were largely unaffected. Professor McClure has falsely associated these non-procedures with the Lombardi et al. paper as explained by Mikovits et al. in a ‘technical comments’ addendum to the Science paper. Mikovits et al. state that “All samples were blinded, as mandated by the NCI and WPI institutional review board approvals. All experimental procedures were done by the same personnel, in the same physical laboratory space, under identical protocols. Investigators at NCI received 100 samples from individuals without knowing their health status; furthermore, the samples were sent to NCI directly without passing through the WPI laboratory space. Laboratory workers at the NCI and the WPI who performed the polymerase chain reaction (PCR) and immunological studies used coded, blinded samples that did not reveal the CFS status of the individuals.” These technical comments can be found here http://www.sciencemag.org/cgi/content/full/328/5980/825-d

                                                                                                                                                                                                                                 Professor McClure has erroneous information concerning this crucial aspect of the Lombardi et al. study. Contrary to Professor McClure’s assertion, the CFS samples and control samples in the Lombardi et al. study were in fact “blinded, randomised and treated in exactly the same way at the same time.” McClure’s contamination argument seems largely contingent on her erroneous piece of information. With this new information, Professor McClure’s conclusion that the “weight of evidence indicates laboratory contamination” is further jeopardised. If Professor McClure is to maintain her contamination hypothesis, she must now provide a mechanism explaining how contamination occurred in only the CFS sample subset that was part of the larger set of blinded and coded samples that were treated in an identical manner.    

                                                                                                                                                                                                                                Professor McClure is correct in that the Lo et al. study was not blinded or randomized. Dr. Alter has explained that “We did not specifically blind and mix the two sample groups (CFS and blood donors.) However, they were studied in parallel by polymerase chain reaction (PCR).” Dr. Alter’s assertion that the samples were “studied in parallel” is reiterated by the Lo et al. paper itself which states “PBMC DNA (30–40 ng) from the CFS patients and the healthy blood donors, as well as serial dilutions from 50 to 1 fg of mouse DNA mixed with 35 ng of human DNA as the positive templates, were tested in parallel.”Another section of the Lo et al paper says, “All patient and control samples were coded and tested in parallel.” If both the CFS group and control group samples were tested as part of a continuum in a random sample order then the contamination likelihood is very small.

                                                                                                                                                                                                                        Explanatory Power and Mechanisms

I will now examine the evidence for the contamination hypothesis and the rival CFS- XMRV/MLV high correlation hypothesis. The evidence I will present is entirely empirical.

This is the empirical evidence supporting a CFS-XMRV/MLV link:

1. XMRV from patient samples has been seen from a transmission electron microscope.

2. XMRV has been grown from CFS patient’s blood in culture and uninfected cells have been infected with XMRV via CFS patient’s serum.

3. The Lo et al. paper saw the MLV related virus mutate over a 15 year period in seven individuals.

4. XMRV has produced antibodies in CFS patients.

5. XMRV from CFS patient samples has been sequenced and it has been deemed to be distinct from every known murine ERV.

6. The XMRV found in a high percentage of CFS patients is almost identical to the XMRV found in prostate cancer patients.

7. A diversity of MLV’s have been detected in CFS patients. This is consistent with a retroviral infection.

8. Five laboratories have published data showing a high prevalence of XMRV in CFS patients as opposed to controls.

9. These laboratories have found non-ubiquitous and comparable levels of MLV related virus in CFS patients and controls from several studies.

10. Three further studies have found comparable levels (to the other five laboratories) of XMRV in controls. All of these laboratories have found XMRV in a noticeable percentage of controls.

11. Countless contamination tests have come back negative for all studies detecting XMRV in both CFS patients and controls. Some of these tests have even been created by authors of XMRV negative studies. These tests have still failed to detect any traces of contamination in the XMRV positive samples.

12. Hanson et al. have independently confirmed the Lombardi et al. study.

 

The following is the empirical evidence in favour of the contamination hypothesis:

1. Four studies have failed to find XMRV in CFS patients or controls.

 

Based on the above empirical evidence, I will present a hypothesis explaining the “CFS-XMRV/MLV link: hypothesis.” This hypothesis must be consistent with all of the above 13 empirical points of evidence. The cohorts, testing mechanisms (all which were not replications of the Lombardi et al. study) and the blood collection process including type of collection types used were the reason for the “Four studies that have failed to find XMRV in CFS patients or controls.”

 

 Based on the above empirical evidence, I will present a hypothesis explaining the “contamination hypothesis.” I am not a retrovirologist however I cannot conceive of any contamination mechanism that can adequately explain all of the above 13 factors. Each individual point from 1-12 provides an argument against contamination and in order to sufficiently explain all 12 points, multiple contamination sources that have gone undetected by the tests and have coincidentally found comparable XMRV/MLV levels in CFS patients and comparable XMRV/MLV in controls (by 8 laboratories) is required. Also, unique to most contamination occurrences, non ubiquitous levels of XMRV would require an atypical contamination scenario. The antibodies to XMRV in CFS patients would need to indicate that CFS patients have a similar virus to XMRV, but not XMRV. The large diversity of XMRV/MLV detected would require multiple undetected contamination sources. These would be new and distinct sources, unique from every currently known murine ERV. The likelihood of all of these factors occurring simultaneously in multiple laboratories is extremely low and very close to zero. Professor McClure’s comment that “I believe the weight of evidence indicates laboratory contamination” is therefore erroneous. Occam’s razor suggests that “the weight of evidence” significantly supports the CFS-XMRV/MLV connection.

 

Conclusion

Professor McClure has used numerous fallacies, logical inconsistencies, explicit contradictions and flawed arguments to support her conclusion that contamination is the most likely explanation for the CFS-XMRV/MLV results. I have demonstrated that some of these arguments that Professor McClure has presented to support the contamination conclusion, inadvertently provide more weight against the contamination hypothesis. Contrary to Professor McClure’s claims, I have demonstrated that the weight of evidence strongly supports the XMRV/MLV- CFS link arising due to a pure retroviral-illness correlation as opposed to contamination.  

 

 

 

 

 

 

 

Advertisements