A ‘Draw’ at the Blood Working Group Meeting

Posted by Cort Johnson

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A Wild Day- the Blood Working Group Reports – All thanks have to go to ValB626 on the MECFS Forums for just pouring out page after page of details from the hours of testimony and discussion on XMRV. Without it we would have had very little.

It was exciting, we learned quite a bit but unfortunately little resolution was in sight and in some ways the two sides seem to have dug in deeper. It seems clear, for instance, that Dr. Stoye has made up his mind. Dr. Coffin seems to be more concerned about the possibility of contamination than before and probed Alter/Lo quite strongly but is waiting for more evidence before he makes up his mind. On the other hand Dr. Lo and Alter seemed very confident in their findings and asserted they have done the most sensitive tests for contamination. (Dr. Hansen later reported that she was considering switching to the Lo/Alter mtDNA test). Dr. Lo ended with a strong defense of his findings (and a little swipe at the ‘anecdotal’ evidence presented by Stoye) and with what appeared to be an empassioned appeal for more research into CFS. (He seems to be yet another figure who probably wouldn’t have come near CFS with a 10-foot pole before XMRV but has gotten hooked just as Ruscetti did, and Houghton on the CFSAC panel (another Lasker winner) and now Dr. Lipkin, it appears.

The Test that Binds – What Coffin and others would really like to see from Drs. Lo/Alter is a live virus and/or evidence of p MLV integration into human DNA. Interestingly, the way it was worded on Vals posts Coffin didn’t seem to expect that this would have happened yet (“No virus to go with those sequences, as yet?“..rather than “So you still can’t isolate the virus, huh?”). The WPI’s ability to isolate the virus was a key factor in convincing the research world XMRV was real and its apparently not an easy task.

Coffin has been working on determining just what is XMRV and what is not and has developed a test that can distinguish XMRV sequences from mouse sequences. We got something of an idea where he was coming from when he stated that two papers (authored by Dr. Huber and Dr. McClure?) will soon appear that determine what looked like XMRV in CFS patients was actually mouse DNA. (There was also a presentation at the Workshop that was not published which related how one researcher thought she found XMRV but later determined it was contamination . The kicker there is that my understanding is that she used mtDNA to check for contamination, which, of course, Alter and Lo and the WPI did….so it doesn’t appear that that study would bear on their findings.)

Mt DNA Not Enough Coffin does not think mtDNA is sensitive enough but Lo disagreed and, in fact said their test was more sensitive than Coffin’s. (Love the black/whiteness of this field – the sheer clarity of it – it’s so inspiring…This is why people go into Science) Lo also said that mouse DNA would break up when they amplify it and they had clean, clear sequences. Lo then said even when they subcloned the sequences – they were exactly the same. Coffin then went into all the possible sources of contamination. Lo agreed but stated they put in many negatives which appears to mean they prepared many samples that were negative and then tested them……suggesting that if some mouse DNA had been floating around it could not have come from their lab.

Alter Jumps In – Then Alter apparently jumped in from the audience and provided a very strong point when he noted that they tested the old samples and did new draws and found the same gene sequences which leads to the question how do you get the same contaminant twice 15 years apart? What are the chances of that? They are using different reagants, different sampling techniques, maybe different tubes, they are processing the sample immediately – yet the same contaminant shows up…..That would be incredible. (The whole thing just so reminds me of the Left and the Right in the States; the Right attacks, the left defends…..the Left attacks, the Right moves onto something else…)

The Limits of Detection – Hansen’s presentation emphasized how difficult this pathogen is to find. She did check for mtDNA contamination, had blinded samples and tried several different assays. She found pMLV, just as Alter/Lo did. (Stoye and Coffin emphasized that pMLV’s are quite different from XMRV. (A retrovirologist I talked to agreed; the sequences Alter/Lo found are much closer to endogenous retroviruses than XMRV is; he would have expected the opposite; that the Alter/Lo ‘sequences’ would have clustered around it.)

Still no XMRV – XMRV has a really distinctive region that she searched for but could not find. She did report that she thought the negative studies were caused by using the wrong primers and that they are missing the pMLV’s as well because they are searching too narrowly.

The most astonishing part of her presentation was when she reported that two different PCR machines in the same lab tested differently; one could find it and the other could not. She also had to do multiple ‘transfers’ (rounds?) of PCR to pick it up. We will hear about the ‘limits of detection’ several times during the talk. Could it’s rarity in PBMC’s be what is stimying the efforts? Possibly so…

Some Recovery is Possible Without Antiretrovirals – Interestingly she found the same prevalence of XMRV in ‘severe’ CFS (POTS, etc.) and ‘recovered’ (still symptomatic but can get around) which indicates anti-retrovirals are not necessary for some recovery in some people. (Dr. Mikovits had noted this before). Coffin again pressed her about finding a complete virus…One gets the feeling that if someone could do that he would relax considerably.

Dr. Mikovits UK study – the UK study is quite impressive and it’s a shame she can’t get it published. They shipped samples to the National Cancer Institute, which, of course, has – impeccable credentials and they used a lab there that had never tested for XMRV. They also used two independent labs and everything was blinded. They cultured the samples for from three to six weeks, far longer than other studies have tried….. It was a great cohort, all Canadian Criteria, 50% homebound…if XMRV was going to show up in any patients it was going to show up in these – and it did – 78% were positive for XMRV and 4% of controls (as in the first study).

Coffin, interestingly, seemed to back off a bit. After suggesting some problem with the DERSE procedure Ruscetti jumped in from the audience to note the consistent results from both the gag and env regions.

Conclusions –A Draw! Both ‘sides’ drew a little blood and both will come back to fight another day. Stoye is stewing about XMRV and believes the pMLV’s are just mouse DNA, Coffin is quite concerned, Drs. Lo and Alter are cooly confident, Ruscetti still can’t believe we’re still at the diagnostic stage, Mikovits popped in with an excellent study, Hanson can find it one PCR and not the other!

Hansen and Lo still can’t find XMRV, Dr. Mikovits is finding it in spades, Hansen says the negative studies using the wrong primers, Mikovits says just culture for a few more weeks, fellas and Stoyes just scowls..the only person we were missing was Dr. McClure (:) who we will apparently hear from soon)…..

Alter/Lo still need to isolate the virus or prove its embedded in human DNA. The BWG seems to have no handle on what lab is ‘right’ and has alot more work to do. Right now best thing to do with the BWG timelines seems to be to double them and then double them again….All in all just another day with XMRV!

We should know more after the CAA’s webinar tomorrow at 1p EST..Hopefully we’ll get another timeline (joy), some translation of the BWG’s findings and a reason why after all these months they only passed around four samples…

Isn’t it time for Singh autopsy study? :)

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