XMRV and Culturing, HERV's and more

[ukxmrv]

I
Maybe it does not matter to you but I think that would matter a lot to the many labs who have spent hundreds of thousands of dollars of their budgets running tests based on the Science paper. What is published peer review like this must be adequate information. These labs, particularly private ones but also University, are all competitors. We do not know their real relationships of these labs behind the scenes. Anyway, finding a specified virus in a specified population is a very generic finding, labs should be able to validate that on their own given the information in a journal article. If Mikovitzs has to give 'extra' information behind the scenes, then the Lombardi et al article was incomplete.

I am glad that you brought up the scandelous waste of money by the teams using their own methods for finding XMRV. If there had been one step missed out by WIP and if the WPI had not been willing to share information, then you would have been right.

However, the facts do not support this argument.

The wastage of respources is important to every patient. The money needs to be in the right hands and not in the hands of teams who designed studies that were not even attempts to replicate the WPI finding from the very start.

The labs who attempted to find XMRV did not follow the protocol in even the most basic way. They did not use a similar cohort, they did not use the same methods as per the Science paper and they included researchers who may have a vested interest in a negative coutcome.

They wasted precious funding in a series of repeat errors.

Is it a conspiracy or just plain incompetence. I don't know.

We are sick not stupid.

 

[Gerwyn]

I have been told the importance of amplification varies with the sensitivity of the test. For a less sensitive PCR more amplification prior to testing may be critical, as Gerwyn says, getting the cells to replicate the virus. However, some of the other studies after WPI used more sensitive real-time PCR tests, I believe one of the UK studies mentioned their test could detect a single virus. Therefore amplification might be less critical, as long as DNA was properly extracted.

No doubt more of the pending studies will be using more sensitive tests than the WPI version, which was a standard PCR. Certainly the researchers involved all understand the concentration/sensitivity issue, I don't think that is the only problem here.



Yes, time will tell. Meanwhile the WPI hypothesis is also still unconfirmed information. Just pointing that out, the caution about getting people's hopes up or dashing them goes both ways.

If you want to know about the HERV K18 superantigen in CFS, you might read some of Dr Bridgette Huber's work. That can be activated apparently by HHV, and I have read that HERVs are also more like to activate when methylation is failing (a methylation stage is involved in keeping HERVs quiet).




This is what I was trying to say above. The European study authors probably thought their tests were sensitive enough, at the level that allows for viral detection. They had the count levels and sensitivity and sample volume information from Science. Figuring if their test might work was basic math, and they obviously thought the tests should have worked. And they DID work on positive controls. But they got no PCR hits, so AT THAT RESOLUTION nothing was detected.



Maybe it does not matter to you but I think that would matter a lot to the many labs who have spent hundreds of thousands of dollars of their budgets running tests based on the Science paper. What is published peer review like this must be adequate information. These labs, particularly private ones but also University, are all competitors. We do not know their real relationships of these labs behind the scenes. Anyway, finding a specified virus in a specified population is a very generic finding, labs should be able to validate that on their own given the information in a journal article. If Mikovitzs has to give 'extra' information behind the scenes, then the Lombardi et al article was incomplete.

I dont know who told you that the importance of amplification varies with the sensitivity of the test "proper" extraction of DNA whatever on earth that means is irrelevant .Amplification is paramount


You would have to extract the RNA from the pMBCs and make copy DNA.Once again you cant detect a latent virus using PCR no matter how sensitive--I know facts can spoil an argument
FOR PCR TO WORK YOU NEED REPLICATING VIRUS.If you look for a non replicating virus in blood you will never find it with PCR

Nothing to do with Herv read study

You need replicating virus for PCR no matter how sensitive the test lok in the blood for as long as you like It is time wasted


No one but a complete idiot would try to figure out if a test worked.A scientist would calibrate the test first against a known positive. It has nothing to do with tinking it is effective enough not for a scientist anyway
It is nothing to do with mathematics basic or otherwise oh and by the way they got the sample volume wrong anyway good maths eh!

Amplifying positive laced controls is childs play It does not mean the test worked

Why do they want more information when they could not be bothered to use the information in the study read said study

If they did not want to waste money they should have followed the instructions Simple really is it not

 

[Cort]

This following comes from the Imperial college study

Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness

Ok what does medical screening mean ---In this context patient history and physical examination by a competent physician .Detectable by blood tests OR patient self reporting This is what taking a patients history means.

Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness

Ok what does medical screening mean ---In this context patient history and physical examination by a competent physician . So Detectable by blood tests OR patient self reporting ..This means that the patients had none of the symptoms reported by the patients in The WPI cohort OR any of the biochemical or immunological annormalities. By McCure,s own introduction they are an entirely different cohort to those investigated by the WPI


DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR

Now this seems an innocuous statement does it not?

The point to realise is that she knew about Mulv and the fact that it was closely related to XMRV.

As a virologist surely she would know that Mulv does not replicate when in the bloodstream.It only replicates in lymphoid tissue,after replication it quickly becomes latent..

So knowing this she attempts to isolate the XMRV DNA directly from the blood by PCR even though she knows that this would not be possible with Mulv

She knows that Mulv lives in lymphoid cells but chose not to examine lymphoid cells for XMRV content at all

To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected.

Ok the concentration of Beta globulin gene is many orders of magnitude higher than the concentration of cDNA would be This proves nothing at all



Received: December 1, 2009; Accepted: December 4, 2009; Published: January 6,

2010

Peer review in 4 days could be as little as one .They invite you to believe that underwent peer review in the period between the two dates.How can anyone anyone review a paper in this time.The Science peer review took months

This still seems innocuous to me. Are you saying that XMRV can only be picked up when its replicating? Aren't lymphoid cells in the blood? IF they didn't concentrate the lymphoid cells and then look for XMRV couldn't they make up for this by using a more sensitive technique?

Sure the review went fast and that looks suspicious but that doesn't mean the paper itself is bad - the fast review doesn't necessarily mean anything about the study - it simply suggests something about the Journal. The paper stands on its own. Sure in a quick review they could have missed something..the question is did they?

I don't get your conclusion that the patients had no symptoms like the WPI cohort or even the lab tests. Most of the abnormalities reported by WPI (RNase L, cytokine upregulation....) are not necessarily signs of a specific illness - they are simply abnormalities. Saying that any abnormal lab test will get you disqualified from a study is going way, way too far in my view. They would have to disqualify everyone who has low cortisol, for instance, and they've done study after study on low cortisol patients. That group found evidence of cytokine activation in one of their studies. Does that mean they can never test those patients again? That interpretation is far too reaching for me.

Again, my question which no one has answered or rather you have not answered - is there any evidence in the Science paper that WPI researchers activated cells prior to testing them for PCR. I don't see any?

 

[dannybex]

I am glad that you brought up the scandelous waste of money by the teams using their own methods for finding XMRV. If there had been one step missed out by WIP and if the WPI had not been willing to share information, then you would have been right.

However, the facts do not support this argument.

I'm not certain of course, but I think this may be part of what Kurt's referring to -- from the Prohealth webinar/interview with Dr. Mikovits:

"Question: Im confused about something. You say you cant find it very easily by PCR, so you are culturing it. Then how did you find it for the Science paper?

Judy: Well, because I found it in 67%, by PCR, of the patients, but I looked several times, at any given time. I just said it's like an EEG. So I got lucky and found it when the patient was high. Theyll come to the doctor when theyre sick which might mean theyre replicating more WBCs and theres more virus in their WBCs.

Question: So you checked these patients multiple times over time?

Judy: No, I did them all at the same time because they were in the repository. So we collected those samples multiple times over time.

Question: So you took like 10 samples from one patient?

Judy: No, usually it was four. A Poisson distribution, the copy number could be as low as 5 or 10 copies per mil of blood, so theres a statistic called Poisson distribution, you might find it one out of three times so we went more than one and in most people we found it.

Question: So the UK paper had studied patients. If they would have looked at 4 or 5 per patient, do you think they might have found it?

Judy: They might have certainly found more, yeah."


Correct me if I'm wrong, but it's my foggy-brained recollection that the need to collect or test at least four samples in order to come up with a positive, was not reported in the Science study.

Is that a critical piece of information that wasn't in the original study?

???

 

[Cort]

That was not in the original study. If they did need to activate/culture cells in order to find the virus and didn't mention it that would be a spectacular 'boner' and I just can't imagine that that would happen. I assume that every author of the paper - looked at it closely - and as we know these were no lightweights - some of them had long resumes - I can't imagine they would have missed that. The best explanation for me is that they didn't do it.

 

[Esther12]

re studying multiple samples: Could this have just been left unclear if the Science paper talked about 'patient samples' (plural) but made no specific mention of taking multiple samples from the same patient?

I remember at the time hearing that Science were apparently uninterested in publishing lots of additional details with this paper. I can't remember where I read that though, and it sounds a bit strange now I repeat it.

 

[julius]

Does anyone remember, and I think it was in the same interview, when Dr M said that there were many more details they wanted to give, but science edited them down? And I'm not talking about wanting them to not mention CFS, I mean technical details.

 

[kurt]

Hey Kurt. I am wondering how the HERV theory squares with the statement by Dr Mikovits that they sequenced the entire XMRV genome two and a half times. It seems to me that with the whole genome you would know for certain if you were looking at a HERV or XMRV.

yes you would I have looked at the sequencing a herve would stand out like a sore thumb

Correct, the sequencing would distinguish the type of antigen very well. However, the lab where the sequencing study was probably sent (Silverman's) had previously conducted extensive studies on that exact same XMRV antigen. So if there was a false result, contamination is one possible explanation for the sequencing study.

Each test in the Science article has to be evaluated separately, if the study failed there would have to be an explanation for every part. But there could be multiple problems involved. MuLV cross-reaction for antibody, culture and antigen studies, contamination perhaps for sequencing study. I don't know of an alternate explanation for the PCR, but false positives can have many different explanations.

The purpose of external validation studies is to sort through all this, and verify that WPI found what they said they found, using alternate test designs where possible (which strengthens the case). So far all we have seen is a few PCR tests that could not find XMRV, and one antibody test that appeared to find something else in controls. So jury is still out.

 

[Gerwyn]

This still seems innocuous to me. Are you saying that XMRV can only be picked up when its replicating? Aren't lymphoid cells in the blood? IF they didn't concentrate the lymphoid cells and then look for XMRV couldn't they make up for this by using a more sensitive technique?

Sure the review went fast and that looks suspicious but that doesn't mean the paper itself is bad - the fast review doesn't necessarily mean anything about the study - it simply suggests something about the Journal. The paper stands on its own. Sure in a quick review they could have missed something..the question is did they?

pcr only works if virus is replicating----well known fact

lymphoid cells are white blood cells

No you cant make up for it with a more sensitive technique This seems to be a frequently quoted misconception

i think i explained medical screening if you dont accept my explanation the please feel free to look up the meaning

It may seem inocuous to you but as a fact it is not

If you screen out all medical abnormalities then you have a patient group with no medical abnormalities when subjectively or objectively measured

The WPI cohort had medical abnormalities both when objectively measured and sunjectively measured.

As the Mcclure cohort had no dectectable medical abnormalities that means that means that they did not report any symptoms that could be attributed to organic illness or have abnormal blood parameters of any kind again please check the processes involved in medical screening if you still doubt that

As the WPI cohort were characterised according to the Canadian criterea they reported symptoms that could be attributed to organic disease and they had objectively measureable abnormal blood parameters.

i am sorry that you think that this objective fact is going way too far There is no need to interprate facts the selection criterea are clear enough

I suggest you have another look at the conditions the patients that the wpi patients reported - by the way believe it or not measuring abnormalities in the blood is a major method of diagnosing organic disease.unless of course they have rewritten the medical textbooks

 

[kurt]

A technical note for Gerwyn and others. For the record, to include a quote in your post, you must have both the quote start at the beginning (that is the word 'Quote' in brackets [] which may include the person's name you are quoting but that is optional) and quote end statements (that is the word '/Quote' also in brackets []) at the end of the quote. That can be just written in the regular message text.

This would be helpful because otherwise it is hard to know where the quote ends and your comments start.

 

[Gerwyn]

origially posted by Kurt

Correct, the sequencing would distinguish the type of antigen very well. However, the lab where the sequencing study was probably sent (Silverman's) had previously conducted extensive studies on that exact same XMRV antigen. So if there was a false result, contamination is one possible explanation for the sequencing study.

now!!Since when is a Herv an antigen?The sequencing was done on the xmrv genome.
Where the sample was PROBABLY sent .So you dont know where the samples were sent."So IF there was a false result."There is absolutely no evidence that the result was false.Contamination is one possible cause--so contamination could take a Herv sequence out that would indeed be a first

Each test in the Science article has to be evaluated separately, if the study failed there would have to be an explanation for every part.

But there could be multiple problems involved. MuLV cross-reaction for antibody, culture and antigen studies, contamination perhaps for sequencing study.

I don't know of an alternate explanation for the PCR, but false positives can have many different explanations.

Mulv cross reaction has been ruled out repeatedly "false positives can have many explanations " name one that could be possible in the WPI study

Each test has to be evaluated seperately says who?

The tests used in the study are used precisely because they have been validated

The purpose of external validation studies is to sort through all this, and verify that WPI found what they said they found, using alternate test designs where possible (which strengthens the case). So far all we have seen is a few PCR tests that could not find XMRV, and one antibody test that appeared to find something else in controls. So jury is still out.

when are alternate test designs possible? How does using an unproven assay method strenghen any case?
if you want to validate a test you cant use a different test
Everyone in the field of retrovirology including Groom and McClure accepts that the WPI isolated XMRV---Are you really suggesting otherwise .Are you putting your lay opinion ahead of theirs?

 

[julius]

So if there was a false result, contamination is one possible explanation for the sequencing study.

The XMRV that WPI was looking at is said to be different from the prostate one Lombardi was studying. Could someone here have a look at the two complete genomes (if Lombardi did one?) and see if they are the same or different?

 

[natasa778]

Re the need to collect multiple samples (at different times) in order to 'hit' on the active period of the virus... that was exactly what was suggested should be done/considered in Q&A session following abbots diagnostics guy CROIT presentation - they were puzzled as they were getting so few positives through antibody tests (just developed xmrv-specific antibodies). Although they were looking at healthy blood donors they only got about 4-5 clear positives if I remember correctly out of hundreds of samples (was it 800 or something like that?).

Then someone suggested it would be interesting to retest the same cohort at different times to check if this could be down to viral activity phases....

 

[Knackered]

Post deleted by moderator - insulting to another forum member.

 

[julius]

And it's always great to read your insightful, intelligent posts as well knackered.

 

[Knackered]

And it's always great to read your insightful, intelligent posts as well knackered.

It's just a bit of fun.

 

[Jody]

No Knackered, it's not fun and it's not going to be tolerated.

No more of these types of posts please.

 

[Gerwyn]

The XMRV that WPI was looking at is said to be different from the prostate one Lombardi was studying. Could someone here have a look at the two complete genomes (if Lombardi did one?) and see if they are the same or different?

98.6% similarity

 

[Knackered]

No Knackered, it's not fun and it's not going to be tolerated.

No more of these types of posts please.

It's not a barrel of laughs having this illness either Jody as you know. Being berated by seemingly vitriolic posts about the only valid cause about my illness I've ever heard isn't much fun either.

I appologise, but it's just a joke, no harm intended.

 

[julius]

98.6% similarity

Yeah, so if one of you sciency guys could take a look at the complete sequence the WPI did, and compare it with a complete sequence by Lombardi (if he did one), we could see whether it is a contamination issue.

And before anybody jumps on me, I know that there wasn't contamination in the WPI lab, but there is the possibility of it in Lombardi's.