TWiV 164: Six steps forward, four steps back

Your review of novel retroviruses is missing several crucial pieces of information.

Mikovits and Ruscetti sequenced polytropic gag regions of a retrovirus present in people diagnosed according to the Canadian definition of ME.  

Isolates of these viruses were sent to Silverman at the Cleveland clinic who instead of sequencing the viruses sent to him, sequenced the VP62 plasmid contaminant present in his lab.  This plasmid and all the cell lines contaminated with VP62/XMRV were never in the WPI/Mikvoits, NCI/Ruscetti, FDA/Lo or NIH/Alter labs.  This was later discovered and that data alone was retraction from Lombardi et al.  In other words the data that determined the tropism of the viruses was no longer present, but the other nucleic acid data from Mikvoits and Ruscetti was still present.  It is therefore wrong to suggest the paper was gutted. 

The remaining experiments still stood.  Nested PCR for gag sequences from LNCaP cells that have been co-cultured with subject’s plasma or activated PBMCs.  Presence of antibodies to XMRV Env in subject’s plasma.  Presence of gag products by nested PCR on stimulated PBMCs or detection of viral proteins expressed by activated PBMCs with appropriate antisera.  Nested RT-PCR of plasma nucleic acid or PCR from cDNA from unactivated PBMCs.  PCR of DNA from unactivated PBMC prepared from subject’s blood.

Since then new env sequences were added to the genbank revealing the viruses to be entirely polytropic.  A match with the Lo et al. viruses.  

Studies that have only used VP62 to set their assays to, which is a synthetic form of XMRV never found in nature and never once claimed to have been found in prostate cancer or ME patients, have not looked for the XMRV in prostate cancer patients (which is represented by VP42) or the Polytropic viruses found in ME patients.  Some papers claimed to be negative for XMRV in prostate cancer were also positive.

During this show there are claims made that retroviruses do not integrate into the same nucleotide position.  This is untrue and it has been found that both MLVs and HTLV do this.  This can easily be found by searching the published literature.  

Claims are also made around the use of certain contaminated PCR kits, but these are not the kits used and those are contaminated with MLVs, which are mouse viruses.  

The paper Paprotka et al is also highlighted, though this paper has not been replicated and contains no real substance.  The later xenografts in this paper were not shown to be from the same cell line or contamination free, the assays used were never shown capable of detecting less than 2000 copies per 100 cells, and a third assay said by Pathak to be an reverse transcriptase PCR assay is omitted from the paper.  Unless a person happened to have watched and remembered the CROI conference they are left to guess that this assay was used and then guess at what conditions were used as nothing is recorded in the paper.  Science should be investigating this as they do require all details to be published.  As we all now know the this cell line does not contain the viruses found in ME patients or the XMRV found in prostate cancer it doesn’t matter when they claim VP62/XMRV was accidentally made, and miraculously didn’t infect humans (92-93).  There is also the 293T cells that are also infected and date from 1977.  You cannot prove they did not infect 22Rv1, or that that patient was not infected, nor can you prove another cell line didn’t infect 293T, or that a human didn’t infected 293T.  The qualitative PCR assay used in Paprotka also failed to detect virus in 293T cells.  Not surprising when they were never shown to find less than 2000 copies per 100 cells.

The blood working group study is equally fascinating.  Using assays not proven to detect the viruses, they didn’t detect the viruses.  These assay also were set for VP62, again not PMRVs.  The Lo team used the failed assay from Lo et al., not the successful assay.  The WPI (Lombardi) changed assays.  The contamination was also not XMRV or MLV contamination, it was mycoplasma, and the blood group refused to let them complete culturing.  The controls in the study could also not have been deemed negative for the viruses because no PBMCs were sent out for pre-screening and not all labs received them to pre-screen.  

Interestingly Annette Whittemore has said the test used at ViPDx by Lombardi, who did the testing for the blood study, was not the test used by the clinical lab.  And if you read the blood study and the Lombardi paper you can see the assays are completely different.  

Neither Dr Frank Ruscetti, Dr Sandra Ruscetti or Dr Judy Mikovits agreed to retract.  They all explained that the viruses are polytorpic.  The gel from the paper was seen during peer review and Science had the labels changed to protect patient identities and to simplify the experiment for publication because hundreds of western blots had been performed.  Science and the reviewer knew about the use of AZA as they saw the oriental gel and patients can been found discussing the use of AZA two years ago.  John Coffin was also at the CFSAC three weeks after publication where Peterson discussed the details of those assays and again the use of AZA.  Again there were no complaints then.  It therefore makes no difference in the conclusion and neither the Ruscetti’s or Mikvoits have ever stated it does.  It was a blinded study and not knowing what would be needed to find the virus different tests had to be run.  A single gel would never provide this information, but was used to illustrate the bare bones, as approved and requested by Science.  

So in summary no replication has been attempted of the proven methodology in either paper, there is no evidence of contamination in their work or samples, as confirmed by the CDC retesting 20 positives.  

In relation to the Lo paper those authors have also stated they stand by the integrity of the data, regardless of if they have been persuaded to retract for political reasons, as again no scientific evidence has been forthcoming to refute the findings and they do confirm the Lombardi findings.  No other lab replicated, sample numbers are irrelevant as more can be obtained and from the original patients.  They have also published nothing new on not been able to detect the viruses but the blood study in which thy used the failed assay from the Lo paper, not the proven assay, so that is not replication.  So yes it is a weird retraction as is the Lombardi paper retraction by Alberts. 

Why would people’s careers be riding on this?  And who has the power to authorise what gets researched and why?  Perhaps central control should be disbanded for control by a few can only hinder scientific progress.  A single study can also not determine by itself why results are positive.   Suggesting this is the antithesis of science.

Ah. Ok. Found it: http://projectreporter.nih.gov/project_info_description.cfm?aid=8263583&icde=10870370&ddparam=&ddvalue=&ddsub=

Sean,

I’m
not going to enter into a debate on this but here are just a few errors in your
logic/claims:

1)  “Mikovits and Ruscetti sequenced polytropic
gag regions of a retrovirus present in people diagnosed according to the
Canadian definition of ME.”

They
sequenced *something*.  However, without
showing integration sites, they may very well have been (and probably were)
sequencing contaminants.  Sequencing bits
of something is not evidence of a true novel retroviral infection.  You have to show that it actually integrated,
especially if the possibility of contamination is as high as it is here.

2) “This was later
discovered and that data alone was retraction from Lombardi et al.  In
other words the data that determined the tropism of the viruses was no longer
present, but the other nucleic acid data from Mikvoits and Ruscetti was still
present.  It is therefore wrong to suggest the paper was gutted. “

The
rest of the paper was also retracted due to the lack of proper controls,
without which, their data is meaningless.  The paper was in fact gutted.

3) “Since then new env
sequences were added to the genbank revealing the viruses to be entirely
polytropic.  A match with the Lo et al. viruses.”

Lo’s
paper was strongly indicative of contamination as well which is part of the reason that they
retracted it voluntarily.  Lo’s
retraction further states:

“…in
consideration of the aggregate data from our own laboratory

and
that of others, it is our current view that the association

of
murine gamma retroviruses with CFS has not withstood the

test
of time or of independent verification and that this association

is
now tenuous.  Therefore, we retract the
conclusions in

our article.”

Thus, even they
accept that the XMRV-CFS hypothesis is crap and that their paper’s conclusions
are unreliable.

4) “During
this show there are claims made that retroviruses do not integrate into the
same nucleotide position.  This is untrue and it has been found that both
MLVs and HTLV do this.  This can easily be found by searching the
published literature.”

Reference?  They may insert in similar regions but
insertion into identical locations on the chromosome is indicative of
contamination, not true insertion.

5) “Claims are also
made around the use of certain contaminated PCR kits, but these are not the
kits used and those are contaminated with MLVs, which are mouse viruses.”

 SuperScript Reverse Transcriptase was one of
the reagents found to be contaminated. 
This was the same RT used by Lo in their nested RT-PCR.
6) “The paper Paprotka
et al is also highlighted, though this paper has not been replicated and
contains no real substance.  The later xenografts in this paper were not
shown to be from the same cell line”

Yes
they were.  They were previously
genotypes in another paper (see http://onlinelibrary.wiley.com/doi/10.1002/pros.10290/abstract).  I’ve seen this brought up on the Me/CFS for a
before, so I’m not sure how you couldn’t know about it.

 Ect…  
in other words, your claims have no merit.  I won’t go into the rest of your post but
rest assured that it is equally erroneous. 

Are you, Alan Dove and Prof. Racaniello, saying you think Mikovits and/or others on the Lombardi paper lied about the results or blinding?  I think circumspection is a natural human reaction to the allegations of theft that have been made against Dr. Mikovits  (my impression is that she was at least out of line, maybe worse, but I think we need to wait for all the evidence in those cases to come out before we make final conclusions).

And this rigorous study by Lipkin won’t be believed by you, you say, if the results confirm the Lombardi paper- the burden is on them, not the other less rigorous studies.

You guys do make some good points.  There are multiple lines of evidence against XMRV infecting humans in vivo.  However, your assumptions and conclusions about the “pro-HGRV” scientists seem to me to be biased once again as compare to your conclusions about the “anti-HGRV” scientists and your silence on the outright fraudulent ‘scientists’ involved in XMRV and in ME science in general- eg CDC, Wessely school including McClure. 

There is some circumstantial evidence of potential ‘sketchiness’ re Mikovits and you have no problem assuming the worst (which is a normal human reaction), but why the double standard when it comes to the “other side” – the proven frauds who wage war on ME science?  I have asked this numerous times and don’t get an answer. 

I appreciate that you published David Tuller’s piece on CDC, but that’s all you’ve done.  In this podcast you mentioned that he wrote the piece but you didn’t mention that it’s another documentation of the fake science done on ME by CDC.  This is bias; I can’t think of another way to explain it.

Wow. Sean Kell makes a number of detailed points that he claims have been absent from the review, a couple of which Poodle Stomper contests. From this, Poodle Stomper grandly claims “etc. in other words, your claims have no merit. I won’t go into the rest of your post but rest assured that it is equally erroneous”. Such a ludicrously grand and prejudicial claim demonstrates Poodle Stomper is writing with a rhetorical agenda, rather than any clear knowledge of the science, and what is more, that rhetorical agenda is to close the door on discussion of the EXTREMELY thorny scientific AND political issues thrown up, not only in these two retractions, but in the response to the possible retroviral link in ME/CFS. It makes Poodle Stomper a highly unreliable narrator.
What we have been seeing, since the Lombardi paper was first published, is a concerted, sustained attempt to ‘shut the door’ on possible retrovirus-ME/CFS link research. The constant claims that the ‘science is done’, ‘the link is dead’ ‘the science is finished’ are shocking, coming as they do from people claiming scientific expertise – that sort of thinking is what some wags would call the ‘schoolboy error’. The idea that Lipkin’s study would be ‘definitive’ is another example of that error of thinking. In the meantime, the poor, besieged patients and their supporters, the ones misrepresented as ignorant, irrational, hostile recalcitrants, time and time again on the so-called ‘science’ blogs, the lazy newspaper and science journal accounts, continue to raise rational and science-based concerns: The lack of actual replication of the original research; the problem of the cloned viruses; the problems around discrepant cohorts used by those promoting psychogenic dismissal of a serious illness acknowledged as neurological by the World Health Organisation; the whole context of ongoing, fallacious psychogenic dismissal of multi-system dysfunction; that antibodies to retroviruses don’t just manifest in ‘contaminated’ blood samples already removed from the body and stored in refrigerators; the evidence suggesting there might be another retrovirus to be looked for even if ‘XMRV’ didn’t pan out; that ME sufferers are still prevented from donating after their death ‘for the good of their own health’. These are just a few discrepancies I can think of, off the top of my head. There are many more.In this context, patient advocate Khaly Castle’s comment is still relevant (and sadly likely to be for the foreseeable future): ”Patients are not pushing for a favorite pathogen. Patients are pushing for real science to occur and to take its course before the door gets slammed on ANY potential avenue of study. Patients are not stupid and are tired of being treated as such. Patients are particularly irked that they point out the discrepancies in scientists’and government’s claims, and said scientists and government continue to push the mistruths forward as if by saying it loud enough and long enough, it will be true.”

The fact of the matter is that there are so many errors in the post of “SeanKnell” that it would take an enormous amount of space to address them all (with the result of noone else reading it). Like the saying goes, a fool can ask more questions that seven wise men can answer.

Because I am no wise man but another fool, I will still have a go at it:

- The WPI’s partial gag sequences uploaded to Genbank are still (essentially) identical to Silverman’s VP62. This means it is almost certain that WPI still contaminated their samples, and that other studies that used primers targetting XMRV/VP62 gag, were not searching for the wrong thing.

- Three argument are in Silverman’s favor: 1) Silverman rigorously controlled for contamination (see the Retraction SOM). 2) Silverman handled all samples in exactly the same manner from start to finish (the WPI did not), which is an excellent control against selective contamination of only one set of samples. 3) Silverman only performed single round PCR , which is much less prone to contamination than nested PCR, which requires the opening of tubes mid-experiment. WPI performed nested PCR.

Conclusion: while it is of course theoretically possible that Silverman selectively contaminated his samples with essentially exactly the same (partial) sequence that the WPI first detected in those samples (!) , it is extremely unlikely.

- Mikovits saying that she didn’t detect contamination in those master samples doesn’t say much, because: 1) she refused to share her data so it’s impossible to check on the validity of the data, and 2) the samples could have easily gotten contaminated at a later time, i.e. after WPI seperated the sample that was sent to Silverman from the master sample.

- The VP62 plasmid was certainly in the WPI labs. Mikovits has confirmed this and there are even experiments involving spiking stuff with some good old VP62 in the original study. This is again some factoid “SeanKnell” (and V99/Gerwyn) has to hang on, despite the overwhelming evidence to the contrary.

- The experiments from the Lombardi paper stood until the paper got retracted for three reasons, which were even in isoltaion sufficient to warrant a full retraction: 1) there had been contamination in at least one of the labs (this lead to the partial retraction). 2) The authors misrepresented a figure and 3) the authors were unable to replicate their results on samples that were chosen by THEMSELVES, controls that were checked by THEMSELVES and assays that were chosen by THEMSELVES.

- Papers don’t use VP62 to “set” their assyas to. They use it as a positive control. Other circumstances (like primers/PCR master mix) determine what you have “set” your assays to. The use of VP62 is perfectly acceptable in this context, and you have only set up this strawman to relieve you of the immense cognitive disssonance caused by all those negative studies. By arguing “all VP62 are worthless”, it makes it possible to discard them all.

By the way, there are no bona fide positive controls. Not one person exists on this planet that we can draw blood from and we (or Mikovits) know must be positive. No, sometimes they are and sometimes they are not. Even replicate positive samples are sometimes positive and sometimes not.

- Like Poodle Stomper explained, the check on the late Paprotka samples had already been done. But regardless, it’s not a very good argument that is being used by you, V99 and Gerwyn.

Consider this: did Ruscetti and Mikovits perform short tandem repeat analysis to verify that their patients and controls were actually the same? No. Is that a bad thing? Again no. While it would eliminate a tiny chance of someone having mixed up the samples, you are really working with scarce resources and must simply choose between the marginal reward of each experiment. Mixing up samples is highly unlikely to have happened, it’s excessive to demand performing a STR on these samples. 

The same applies to Paprotka’s check. The fact of the matter is that experimental matrerial (like the early xenografs from the paper) have been mixed up before in the past. That is why they did that check with the early xenografts. With the more recent material, there was already no (realistic) doubt these were somehow mixed up. 

In any case (and this is how science really goes), if anyone doubts the origin of those late samples anyway, one could do the analysis themselves and prove everybody wrong. However, sitting by the sidelines, mereley hypothesizing about some miracle event that might have happened, is not the way to go with things like this. At this stage, the onus is on you to prove the authors wrong, and it would be extremely easy to do so if they actually were.

- There is no evidence that Pathak “omitted” an assay from the paper in the sense that you are arguing, which is why the IMEA letter to Science did only get laughed at.

- Your “logic” about  293T is horrible. I could pour an 1993 wine in a 293T cell and subsequently detect the contaminant. That would not mean that the 1993 wine was actually produced in 1977 after all. 

- You statements about “gelgate” are totally incorrect. The original gels were not subject to peer review, no author ever presented information about using 5-aza in the context of figure 2C of the paper before the Science investigation, and even the patent sumbission by Mikovits and Ruscetti omitted this information about 5-aza. As you will know, patent boards don’t have peer reviewers that ask you to leave out data, and require you to submit ALL (and I mean REALLY ALL) relevant information.

- The Blood Working Group was indeed very fascinating, on that we agree. WPI/Mikovits (Lombardi is not named in the paper or the suplement section at all) and Frank Ruscetti chose their own patients, their own assays, and were totally unable to reproduce their earlier results. There is no evidence that Lo used an assay that did not work, except of course for the fact that he too was unable to reproduce his earlier work. And even without any pedigreeing of negative patients (although they were checked by (i.a.) Ruscetti and Mikovits for PCR, serology and culture), there is no way that you’d find that many positives in a control group (even if all 15 were the sexual partners of ICC ME patients you’d not expect these kinds of results).

- This quote of you is quite important:

“A single study can also not determine by itself why results are positive.”
Very, very true indeed. Now, instead of Lipkin, or the BWG, think about Lombardi. OrLo….However, this study is set up pretty well, and if the original results were, through some miracle, true after all, you would expect Mikovits/Ruscetti (or Lo) to reproduce their original findings. You would expect them to detect this thing better and better throught time, not the other way around. In any case, the inability to (self-)replicate is considered to be a accepted grounds for retraction, and there would be really nothing wrong with Mikovits/Ruscetti finally rejecting their original findings after negative results. Remember, they may revisit and ‘rediscover’ their original finding at any time in the future. That is really the ‘scientific’ check against this sort of thing.* BTWWho even believes a certain “Sean Knell” has suddenly entered the debate and is making these arguments that, while factual and logical totally incorrect, require quite of bit of research into the subject.It is perfectly fine to use an anonymous handle, but why would you switch names each time you post something? It makes you look like a rather dishonest and stupid (because it’s easy to see through) person, frankly.

Totally agree. It doesn’t. Not that I believe they are obligated to but it would have been useful to know – even if they then had changed/improved/amended their e.g. selection protocols for patients at a later date.
Still, only a couple of months to wait. And that link did confirm the budget at least – your tax dollars. There was some confusion over that in the media. So, $1.043m it is then.
And those 150 samples – as well as the controls I suppose – will then form part of the CFS Initiative/Lipkin NGS study. Now that will be interesting I think.

1) “They sequenced *something*.  However, without showing integration sites, they may very well have been (and probably were) sequencing contaminants.  Sequencing bits of something is not evidence of a true novel retroviral infection.  You have to show that it actually integrated, especially if the possibility of contamination is as high as it is here.”

No they sequenced polytopic sequences.  You are unable to understand how this is done. Intergeneration studies are done later.  Several labs have presented evidence at conferences to show integration and the viruses in prostate cancer which are XMRV, have been shown to be integrated.

2) “The rest of the paper was also retracted due to the lack of proper controls,
without which, their data is meaningless.  The paper was in fact gutted.”
The lack proper controls was only in relation to Siverman’s experiments, not those of Mikvoits and Ruscetti.  The paper was not gutted, only full sequencing was removed.  

3) “Lo’s paper was strongly indicative of contamination as well which is part of the reason that they retracted it voluntarily.  Lo’s retraction further states:”

There has never been evidence of contamination in either paper.  No samples, reagents, or other evidence has been provided.  Lo and Alter were told to retract.  They have published no further evidence to argue against their paper, no other paper has replicated or used clinically validated assays, there has been no test of time and they did not wish to retract the data.

4) “Reference?  They may insert in similar regions but
insertion into identical locations on the chromosome is indicative of
contamination, not true insertion.”
Incorrect.  This has never been a sign of contamination.  HTLV and MLVs do the same.  See these papers:
http://www.ncbi.nlm.nih.gov/pubmed/3015420 Selten et al. 1986.http://www.ncbi.nlm.nih.gov/pubmed/2884332 Weinstein et al. 1987
http://www.ncbi.nlm.nih.gov/pubmed/18369476 Meekings et al. 2008

5) “ SuperScript Reverse Transcriptase was one of the reagents found to be contaminated.  This was the same RT used by Lo in their nested RT-PCR.”

It was not THE kit used by Lo and Alter. Not all SuperScript Reverse Transcriptase kits are contaminated.  

6) “Yes they were.  They were previously genotypes in another paper (see http://onlinelibrary.wiley.com….  I’ve seen this brought up on the Me/CFS for a before, so I’m not sure how you couldn’t know about it.”

Incorrect.  The later xenographs used in Paprotka were not shown to be from the same patient and were not shown to not have been contaminated with other cells.  This is a common error found in studies.

The quickest way for you to begin to understand this is reading this section from Wikipedia and then using the references given to confirm.

“Cell line cross-contamination can be a problem for scientists working with cultured cells. Studies suggest anywhere from 15–20% of the time, cells used in experiments have been misidentified or contaminated with another cell line.[4][5][6] Problems with cell line cross-contamination have even been detected in lines from the NCI-60 panel, which are used routinely for drug-screening studies.[7][8] Major cell line repositories, including the American Type Culture Collection (ATCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ), have received cell line submissions from researchers that were misidentified by them.[7][9] Such contamination poses a problem for the quality of research produced using cell culture lines, and the major repositories are now authenticating all cell line submissions.[10] ATCC uses short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines.[11]”

http://en.wikipedia.org/wiki/Cell_culture

As you have not made any other arguments your claims have no merit.  Rest assured that I have shown yours to be erroneous.

Your arguments regarding Paprotka look foolish.

“8) “- Like Poodle Stomper explained, the check on the late Paprotka samples had already been done. But regardless, it’s not a very good argument that is being used by you, V99 and Gerwyn.”

There has been no investigation of Paprotka.  An assay is missing.”
9) “Consider this: did Ruscetti and Mikovits perform short tandem repeat analysis on samples they tested from each other to verify that the used samples were actually from the same patients? No. Is that a bad thing? Again no. While it would eliminate a tiny chance of someone having mixed up the samples, you are really working with scarce resources and must simply choose between the marginal reward of each experiment. As mixing up samples is highly unlikely to have happened, it’s excessive to demand performing a STR on these samples. ”

Why would they need to prove each samples was from the same patient?  The main results of 67% were based on the Nested RT-PCR gag assay.  The two studies are entirely different.  

Paprotka would have to prove VP62/XMRV was created during passage through mice.  They would have to prove the early xenografts are negative and the later positive to support this argument, as no other samples exist.  If either of those samples are not from that patient there is no argument.  They have no evidence.  

10) “The same applies to Paprotka’s check. The fact of the matter is that archived experimental materials (like the early xenografs from the paper) have been mixed up before in the past. That is why they did that check with the early xenografts. With the more recent material, there was already no (realistic) doubt these were somehow mixed up. ”

As samples are always mixed up why do the STR analysis on everything but the later xenografts?  Those samples are 15 years old. 

11) “In any case (and this is how science really goes), if anyone doubts the origin of those late samples anyway, one could do the analysis themselves and prove everybody wrong. However, sitting by the sidelines, mereley hypothesizing about some miracle event that might have happened, is not the way to go with things like this. At this stage, the onus is on you to prove the authors wrong, and it would be extremely easy to do so if they actually were.”
Who has access to those samples and is willing to do this?  Scientists are sitting on the sidelines if they let this slip.  They have in short not proven they are right.  They have provided no evidence in Paprokta.  It is up to them to have tested the samples, unless they already knew they were not from that same patient?

12) “- There is no evidence that Pathak “omitted” an assay from the paper in the sense that you are arguing, which is why the IMEA letter to Science did only get laughed at.”

Incorrect.  Pathak said at CROI it was an assay not listed in the paper and the material available for the only later xenograft that links to 22Rv1 would have to be tested with such an assay.  If Science are not taking this seriously perhaps they should close down.

13) “- Your “logic” about  293T is horrible. I could pour a 1993 wine in a 293T cell and subsequently detect this ‘contaminant’. That would not mean that the 1993 wine was actually produced in 1977 after all. ”

There is no evidence as to when 293T cells because infected.  Prove it was after 1993, but don’t repeat the errors of Paprotka to do it. They didn’t provide any evidence.

14) “- You statements about “gel gate” are totally incorrect. The original gels were not subject to peer review, no author ever presented information about using 5-aza in the context of figure 2C of the paper before the Science investigation, and even the patent submission by Mikovits and Ruscetti omitted this information about 5-aza. As you will know, patent boards don’t have peer reviewers that ask you to leave out data, and require you to submit ALL (and I mean REALLY ALL) relevant information.”

All original material was sent to the reviewer.  Including the gel labelled with AZA.  Science had this altered to simplify for publication and for ethical reasons. The paper and patent both then went through Science for publication.  

15) “- The Blood Working Group was indeed very fascinating, on that we agree. WPI/Mikovits (Lombardi is not named in the paper or the supplement section at all) and Frank Ruscetti chose their own patients, their own assays, and were totally unable to reproduce their earlier results. There is no evidence that Lo used an assay that did not work, except of course for the fact that he too was unable to reproduce his earlier work. And even without any pedigreeing of negative patients (although they were checked by (i.a.) Ruscetti and Mikovits for PCR, serology and culture), there is no way that you’d find that many positives in a control group (even if all 15 were the sexual partners of ICC ME patients you’d not expect these kinds of results).”

Lombardi was still the person who tested the samples at VipDx.  The assay chosen by Lombardi was also not the assay from Lombardi et al.  Mikovits and Ruscetti had nothing to do with patient selection.  The study was not a replication study. 

The assay used in the  The Blood Working Group was not the successful assay from Lo et al.  That assay had never detect any person positive before.  Can you read?  The controls were not identified as negative before blinding as no PBMCs were sent out and only some labs received samples for screening prior to blinding. As the controls could not be said to be negative they can test positive.  Who were the controls? How were they collected?  Did any other lab test them for virus before selecting them for the pre-screening process?

16) “Very, very true indeed. Now, instead of Lipkin, or the BWG, think about Lombardi et al. Or Lo et al…..”

Lombardi and Lo studies both found polytropic MRVs in patients with ME.  The negative studies failed to detect a man made virus in ME patients and never looked for Polytropic MRVs.

17) “However, this study is set up pretty well, and if the original results were, through some miracle, true after all, you would expect Mikovits/Ruscetti (or Lo) to reproduce their original findings. You would expect them to detect this thing better and better throught time, not the other way around. ”

Yes you would expect this is the study is conducted exactly the same way, i.e. replication.  You would not expect better and better results if the patient criteria now used does not guarantee that a CCC patient will be included in the study, or if they are hindered from using their clinically validated assays.

18) “In any case, the inability to (self-)replicate is considered to be a accepted grounds for retraction, and there would be really nothing wrong with Mikovits/Ruscetti finally rejecting their original findings after negative results. Remember, they may revisit and ‘rediscover’ their original finding at any time in the future. That is really the ‘scientific’ check against this sort of thing.”

Only when a study is not replicated. When another lab or they themselves publish work that is a replication study.  A basic scientific principle you now appear to understand? Then the data from the original paper is circumspect.  It does however not lead to a retraction, because retracting such data would not allow others to see what was perhaps done wrong.  In fact it only harms science as a whole and allows the use of politics by those with vested interests.

I don’t think retracted means struck from the record forever – most of the retroviral community interested in this has read it already and I have a copy on my laptop which seems to still be there and have survived the retraction….

Anyway an admirable show from RRM and poodle stomper, hats off to you both. I have a few points to make, and I will not address ever angle of the argument.

A quick search of pubmed for HTLV integration sites and reading through the three papers that SeanKell linked to show quite the opposite of the point you are trying to make – retroviruses DO NOT integrate in identical sites in the host chromosome most of the time – EVER. This I knew already but I was struck by how the evidence you present in fact showed the precise opposite of the point you were trying to make. It looks to me like you’ve only read the abstracts of these articles and mistaken preference in genomic location for absolutely identical integration sites. These are not the same. The degree to which this has been mistaken calls into question anything you write as this is such a fundamental mistake. I am not a retrovirologist which was why I had to go and read some of the literature to clarify this point. I re-iterate: I was amazed at how completely, totally and utterly wrong you are on this point. Using your own sources.

A word on replication. In science, replication means asking the same question and getting the same answer. It does NOT mean using the same brand of Taq polymerase and having the annealing temperature within 0.1 C of the original experiment etc etc. It really doesn’t matter about the miniscule differences between the papers in this field. These differences are far less than those you find in other fields – if you really want to see variability have a look at how flavivirus labs do their antibody neutralization assays. There are more methods than you’ve had hot dinners, yet the fundementals of the results are reproducible using the different assays. There is no reason why XMRV should be a special case. The arguments against XMRV infecting humans are way bigger/better than this.

“VP62 calibration.” This is another first. It is very common, indeed usual, to use something “artificial” as a positive control in ANY experiment, let alone PCR. When I do flow cytometry experiments I use phorbol myristate acetate to active cells as a postive control. Is this in any way relevant to something that might happen in vivo? No. Does it serve to tell me that I remembered to add all the right reagents etc? Yes. PCR positive controls are very commonly things like molecular clones or PCR products cut from gels. There is no “calibration” to the positive control. It tells you that the PCR machine didn’t break down halfway through, or that you didn’t forget to add the primers or something (trust me these things happen). There is no way that the XMRV field is so special you can’t use positive controls like in any other area.

“Blinding of samples in the original Lombardi/Mikovits Science paper.” Another red herring. This really doesn’t matter now – as they couldn’t tell the patients from the controls by whatever assay they wanted to use (like they would use one that doesn’t work when their while future rested on it – *COME ON*). The point is not whether they were blinded, it was the fact that the samples were not prospectively collected and the experimental and control groups were not collected at the same time and in the same way. Whilst on this point: “pre-screening” in the blood working group study. This was unnecessary, as most of the patients selected had already been studied by Lo/Alter or Mikovits and were known positives or negatives. They had been extensively studied, not just “pre-screened.”

I am pleased to note that TRIzol has fallen out of discussion. At least this is some progress.

I accept that funds were misappropriated from the CDC CFS budget. The rest of the statements made here (e.g. by Angela Kennedy as well) about consipracy, suppression of research and Wessley etc are, as far as I can see, purely opinion. I would like to know the evidence these statements are based upon. There are no mistruths being pushed forward about this group of viruses other than by people on blogs such seen here – a really spectacular example of the Dunning-Kruger effect.

Lastly, good has come out of this. The study being coordinated by Ian Lipkin will recruit a reasonable sized cohort of CFS patients and controls with detailed clinical information and well archived samples. This is a big step forward and will enable future hypotheses to be tested rigorously. This is something we all welcome in this controversial area, and this awful disease.

Excellent points.

One noteworthy thing to add:

The argument about identical integration sites is moot (and has been for some time). Fact of the matter is that Silverman, because of the Garson et al. paper’s findings, revisited his earlier samples and CONFIRMED that the two identical integration sites were the result of contamination:

http://jvi.asm.org/content/early/2011/09/21/JVI.05624-11.abstract 

In short:

1. Garson et al. found something “cool” (identical integration sites from two supposedly independent experiments performed in the same lab).
2. They proposed that the explanation for this was “contamination”
3. Silverman tested this verifiable hypothesis through an independent experiment  
4. The evidence gathered from Silverman’s experiment strongly supports Garson et al.’s hypothesis.

It’s actually an excellent example of good science in action. 

It also is a nice example of the “methodology” from the few from the forums completely failing. And I don’t really mean in the sense of Silverman’s results themselves, but in the sense that these few keep clinging to obvious falsehoods. There is apparently too much time/effort/emotion/googling invested in their position to give up after being shown wrong.

And BTW: the funny thing about the “new” (and still unpublished) integration sites, is that they were found by “calibrating” assays to know strains of XMRV, inclusding VP62 and 22Rv1, and not to the polytropic viruses Lo found (and Mikovits also reported on later). 

These results are therefore basically meaningless.

I hope you’re keeping a copy of these posts – they contain quite a bit of detail!

I don’t honestly think it’s worth anyone’s time to argue with Sean”Gerwyn”Kell.  He is terminally dull when it comes to science.  I’ve pointed out the reference where the late xenografts were genotyped and found to be from the same patient as the established cell line and the response he gave was “Incorrect.  The later xenographs used in Paprotka were not shown to be from the same patient and were not shown to not have been contaminated with other cells.”  In other words, he doesn’t accept science that doesn’t say what he wants to hear (which is 99.99% of it).  With his posts consisting of nothing more than errors he is unwilling to correct, I think all we can do is be thankful that he is irrelevant to the scientific process.

Dear Angela Kennedy,

from all I have seen, the name “SeanKell” looks like another so called “sock puppet” to me. He (she?) is posting usually the same BS, something cooked up on the “mecfsforums”. His claims have been take up numerous times and he does not care to provide a hint of evidence for those claims. Why should his claims be taken any serious, when he refuse to provide any sort of evidence?
And I have one question for you: Do you know what XMRV plasmid is?

Kindest regards,
Tony Mach

And Angela, if I may share this observation: Poodle Stomper took up the claims by “SeanKell” and refuted them on the basis of facts, while you accuse him of being political – and at the same time you show not the slightest bit of understanding of the scientific facts discussed.

If Poodle Stoomper (and whoever) were wrong, and “SeanKell” were right, I would be up in arms with you – but that is not how it is. Huffing and puffing about “such a ludicrously grand and prejudicial claim” might bring you bonus points on the “mecfsforums”, but it will not bring you closer to finding the cause of your (our?) disease.

These strains were actually sequenced by Silverman who later found contamination in the samples that he had tested, and these data had already been retracted by the authors before the full retraction. 

In fact, it is exactly the remaining argument used by proponents, that this “faulty” sequencing of a contaminant by Silverman has lead everybody on the wrong track until the partial retraction (on 9/22/11). 

Now, of course this argument does not have any merit considering all the evidence (which I won’t repeat), but these quotes from the Lombardi study do not really provide evidence to that effect.

Donating blood is no problem for the recipient. However if for the donor if you are already very fatigued it is perhaps not the best idea.

Yes, but what you are talking about is POSSIBLE LINKS to ME/CFS, but what is needed is EVIDENCE that these Viruses DO CAUSE ME/CFS, and that is something different – and basically that’s what Racaniello said that was missing.

And there are lots of documented ME/CFS cases that DID NOT START WITH EBV (“glandular fever”). I know for example that Montoya in Stanford is trying hard to find possible links to Herpes-Viruses like EBV, he has some indication that Herpes-Viruses might be involved in some cases, but he is far far from showing any EVIDENCE that Herpes-Viruses CAUSE ME/CFS. These viruses might just act up after another viruses causes the main illness – we don’t know and that’s why we need EVIDENCE.

What evidence that you cannot repeat because it doesn’t exist?

The response to comments that was made by Ruscetti and Mikovits in Science does show the study was blinded.  All testing by the NCI is. 

In the podcast, it was claimed that there is no evidence that these viruses can trigger ‘CFS’.  There is.  It’s pretty universally accepted that they can.  We do not know how.

‘CFS’ is a rather empty diagnosis of ignorance, so I would be very surprised if there was ever a single cause identified for all cases.  That doesn’t mean that there is not clear evidence that ‘CFS’ can be triggered by certain viral infections, and at a much greater rate for some than others.  This is probably the only thing about ‘CFS’ which is uncontentious.  If you want more papers, I could easily dig some up.

…

Vincent: Right. I suspect there might be some infectious triggers – I’m
leaving that open – as I said that’s a hypothesis; there’s no evidence.
So…

Alan: Yeah. It has not been disproven…

Vincent: No…

Alan: But neither has any other hypothesis about that disease.

Vincent: Right.’

Yes, there is. 

But first, before you again commit the “no true Scotsman” fallacy using this sockpuppet (don’t worry, there are always others), let’s agree on the definition of “evidence” in the context of scientific research. I would propose that we use the Wikipedia definition:

‘Scientific evidence is information, such as facts, coupled with principles of inference (the act or process of deriving a conclusion), that make information relevant to the support or negation of a hypothesis.’

Agreed? If you don’t please provide us with your definition of evidence in this context.

Which is irrelevant. Their assay doesn’t distinguish pedigree positives from negatives. What is relevant is their study wasn’t prospective.