From “Annals of Neurology” (brief communication), 4 February, 2011.
Analysis of cerebrospinal fluid in chronic fatigue patients for multiple ubiquitous viruses and XMRV
Steven E. Schutzer MD1,*,
Megan A. Rounds MS2,
Benjamin H. Natelson MD1,3,
David J. Ecker PhD2,
Mark W. Eshoo PhD2
University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Departments of Medicine (SS) and Neurology (BN), Newark, New Jersey 07103
Ibis Biosciences, Inc. Carlsbad, California, USA
Albert Einstein School of Medicine, Bronx NY and Beth Israel Medical Center, Department of Pain Medicine and Palliative Care, New York, NY 10003
Email: Steven E. Schutzer MD (schutzer@…)
*Correspondence: Steven E. Schutzer MD, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Departments of Medicine (SS) and Neurology (BN), Newark, New Jersey 0710
Publication History
Accepted manuscript online: 4 FEB 2011 08:50AM EST
Manuscript Accepted: 24 JAN 2011
Manuscript Revised: 20 JAN 2011
Manuscript Received: 22 DEC 2010
Funded by National Institutes of Health. Grant Numbers: AI088765, AI32247
Abstract
Recent reports showed many patients with chronic fatigue syndrome (CFS) harbor a retrovirus, xenotropic murine leukemia-related virus (XMRV) in blood; other studies could not replicate this finding. A useful next step would be to examine cerebrospinal fluid because in some patients CFS is thought to be a brain disorder. Finding a CNS microbe would have greater significance than in blood because of the integrity of the blood-brain-barrier. We examined, but did not find XMRV, nor multiple common other viruses in cerebrospinal fluid from 43 CFS patients using PCR techniques, suggesting exploration of other causes or pathogenetic mechanisms is warranted.
Link to Annals of Neurology HERE
Copyright © 2011 American Neurological Association
This study looked in cerebrospinal fluid, not blood like everyone who is finding MRV’s in people with ME/CFS.
In case anyone is confused by the choice of words used in the paper, VIPDx does not test cerebrospinal fluid.
No health controls were used is this paper, and there is no evidence that the tests used by the authors would have worked.
“They” have done it again! “They” have set up an experiment designed to fail. They jolly well know by now, like we all do, that PCR techniques are just not appropriate when looking for the elusive XMRV. You have to cultivate it in the right cell lines before even looking for it. It’s probably in the CSF of ME patients but in such tiny amounts that it would be masked by contamination or at least accused of being contaminated. And for PCR, you have to have exactly the right primers (all advice & experimental protocols available free from the WPI, likewise materials, primers etc).
Yes, the methodology involved in this study is totally at odds with that of Lombardi et al. Erlwein et al. (McClure and Wessey study) also failed to reproduce any of the methodology employed by Lombardi et al. Currently no one has even attempted to replicate the ‘Science’ papers methodology. Why?
“They” have done it again! x2. What do they mean by ‘CFS’? It would be more scientific if they used the agreed World Health Organisation definition. That would orientate the reader instead of them wondering what is meant by this pseudonym. Good empirical science is dependent on clear taxonomy – the practice and science of classification – so if they cannot obey this basic law what does it say of the rest of the paper? It’s worthless!
I’m sure the pathological evidence for ME will be found behind that blood-brain barrier, but I doubt an active virus will be culprit. As someone who developed ME after an adult infection of glandular fever, I simply cannot accept as reasonable the proposition that I was actually infected with another virus concurrently which caused ME when as we know early childhood infections of glandular fever are so mild as to be undetectable. No, the simple answer has to be that glandular fever is an example of an infection which the juvenile immune system was designed to see off and which the adult immune system is not without incurring damage to the organism through overreaction or some other mechanism and which damage occurs behind the blood-brain barrier either as immune disregulation or actual pathological injury. Practically all tests for ME which are not based upon psychobabble are predicated on evidence being found outside of the blood-brain barrier and are just as much a waste of time.
Childhood infection with EBV may be mild but it is also lifelong – because the virus integrates into your DNA.
For the majority of people it remains there inactive, kept in check by the immune system. But for the unfortunate few, it can be reactivated by the onset of another viral infection.
With regards to your comments about concurrent viral infection. Once you understand that certain viral infections are lifelong, then you realise that you don’t have to have acquired both at exactly the same time for them to potentially have a subsequent compound effect.
thanks for that patronising comment: individuals such as myself claim that they, having suffered from the acute phase of glandular fever, never made a full recovery and remained fatigued as in the acute phase. If I was tested for the EBV virus, I would be positive, however, my blood would not indicate an ongoing infection, so what are you suggesting is my current viral disease status?
You are incorrect.
People who get HIV and EBV (glandular fever virus) can develop lymphoma.
XMRV has already been shown to reactivate because of EBV too.
‘NF-{kappa}B Activation Stimulates Transcription and Replication of Xenotropic Murine Leukemia Virus-Related Virus in Human B-lineage and Prostate Carcinoma Cells.’ (Sakakibara, J Virology, 2011)
http://www.ncbi.nlm.nih.gov/pubmed/21270144?dopt=Abstract
Therefore the research is most certainly no a waste of time!
Thanks for posting this Tony.
I may be accused of naivity here (again) 🙂 …
But if they are not finding XMRV in CFS sufferers, regardless of criteria, even with PCR techniques (and the CDC did not wholly employ PCR) doesn’t that still tell us something?
What I mean is it seems every study that is published that doesn’t agree with the link suggested by WPI is condemned by the more vocal patient cohorts.
All that money and science out there and yet people still think that if the researchers do not adopt the exact same methods as the WPI, there is some conspiracy.
If just one study proves the WPI right, but uses PCR techniques, I suppose it will be received warmly.
I find it all very strange.
Anyway, it is an interesting way of looking for XMRV, in the spinal fluid. Blood-brain barrier – I was reading about that in your excellent Clinical guide.
Good stuff.
Firestorm,
Once again you demonstrate that you have a poor understanding of the crucially important details involved in “virus hunting”.
Yes, it is perfectly possible that with a properly (CCC)defined patient cohort, correct methodology and with the right PCR primers (correctly calibrated against a positive human control) then a PCR only study could strike lucky and return positive results and that would be welcomed. But it would be a lucky shot in the dark.
In the longer term, an effective PCR based assay would undoubtedly be the desirable method for a screening test.
However, PCR is clearly not the right method for the initial virus hunting stage of the process and that is why traditional co-culturing of blood cells with other receptive cell lines is the obvious way forward.
It’s not just the primers, it’s the sequence diversity of the virus. It’s too big to just go blindly in by using a clone to calibrate an assay. Need to use a positive clinical sample. Then there is the collection and sample preparation. Only Lombardi et al. and Lo et al. have controlled their samples, the negative studies haven’t. We all know the CDC used the wrong tubes in their study. Blood that has already been defrosted once, or not frozen quickly enough. There are so many different types of PCR too i.e. qPCR, Nested PCR, RT-PCR, etc. There is the type of sample you are testing which will make a difference i.e. Whole blood, PBMC’s, plasma, cerebrospinal fluid, etc. Inner sets of primers, outer sets too. There is the section of virus you want to look for i.e. env, gag, pol.
There are hundreds of variables, and I’m only mentioning the simple stuff. It all makes a difference. But with this virus they already know that PCR is working at the limits of its sensitivity, so the best method is culturing. Grow the virus first and isolate it. Don’t be fooled by those who are claiming PCR is the most sensitive. Published research has shown this to be untrue. Methodology is everything. As it is with any virus hunting.
The science method has not been applied by any of the nagative studies. Either they are poor researchers or deliberately choosing to change the variables shown to work.
If you don’t use the correct methods of PCR to look for HIV you won’t find that virus either. PCR isn’t plain old PCR, there are an incredible number of alterations that can be made for any test.
It is time that someone attempted a full replication study of Lombardi et al. Still no one has.
You do not need to have virus in the brain to have brain symptoms.
Ever felt foggy headed and depressed during a bout of flu?
That is because the cytokines (chemicals) produced by an activated immune system disrupt brain chemistry.
I think if people with ME had active virus in the CSF or brain you would see much more dramatic symptoms like the ones which occur in encephalitis or meningitis – and which would lead to an acute and life threatening illness.
An over-active immune system can cause the problems we see in ME
This is reassuring. No-one would want to have virus in the CSF or brain.
It was not reported that the rhesus macaques which were experimentally infected with XMRV had XMRV in nervous tissue, though the spleen, lymph nodes, prostate etc. were recorded as being infected.
PCR in more detail.
http://www.mecfsforums.com/index.php/topic,5382.0/topicseen.html
HIV testing does not rely on PCR alone. An antibody test is also used. PCR is not used as a confirmatory test for HIV.
This is a digest of my posts on the Cerebro-Spinal Fluid paper on a patient forum.
It was a Fukuda cohort of patients, three of which had a cold. We really must start insisting on the CCC, the only criteria that has PEM as a mandatory symptom – PEM being the symptom that sorts the “tired all the time” lot from the PwME.
The “quotes” below are from the paper .pdf
“We used VipDx in the capacity of a service laboratory. The VipDX “XMRV PCR/culture test” is a 2 stage process”
Typically slippery use of language – does it mean they used their protocols, or that the test was carried out at VipDx? In similar vein the fact that they sent them to VIP dx does not mean that VIPdx requested or processed them. VIPdx dont carry out procedures on CSF as it is an unvalidated procedure and they would be in breach of their license conditions. But later we discovered that they had 43 patients, and they sent VIPDx a total of five vials of DNA extracted from their samples.
The first vial had the combined DNA of 23 patients.
The second vial had the combined DNA of 20 patients + 3 negative control extracts.
The third and fourth vial contained distilled water.
The fifth was was spiked was XMRV.
Why do this? Too little DNA from each patient? Too cheap to test them all? Too confident they would all be negative?
more slippery language:
“Using the same kit used in the Lombardi et al paper…”
Lombardi et al needed 8 ml of blood these clowns took 100 microlitres of CSF. When doing lumber punctures for virological analysis the typical volume is about 10 ml. The Lombardi et al methodology and VIPdx approach is validated to detect XMRV in pMBCs isolated from blood, not CSF.
At 100 microlitres of DNA RNA extracted there would have been 9000 cells which would have been 1/10000 th of the amount used by Lombardi et al. It would have taken months of culture even if there were any virus in such a small number of cells to begin with.
Bad Science.
If a person had any of the other viruses they pretended to be looking for in the CSF then they would not have had mildly elevated WBCs they would be seriously ill in hospital!
CSF is normally sterile!!
MuLV-related retroviruses will only replicate in lymphoid tissue. In CSF the upper limit of white blood cells is 9 cells per microlitre.
MuLV-related retroviruses inhabit the vascular endothelial tissue of the brain and may also be found in microglia and astrocytes. They can get past the BBB as though it was not there.
As this paper stands it should not (once again) have got through peer review. Lombardi et al used two different sets of primers and two different kinds of PCR cycling conditions in the Science Paper. Schutzer et al don’t state which they used, hence the paper cannot be reliably replicated and thus should be automatically retracted
I think it very likely that they weren’t careful enough. This study does give the full procedure, unlike the one under discussion, and they are talking about tweaking primers and culturing for 4 – 7 days: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC105134/
This page gives more general information on spinal taps and CSF (so easy to mistype that ) http://www.nlm.nih.gov/medlineplus/e…cle/003769.htm
Once again that the patients in the study we are discussing were not given a formal diagnosis of CFS by a qualified physician
Also neuropathology is typically caused by polytropic MLVs and not xenotropic ones. Looking only for a Xenotropic MRV and not using Lo/Atlers methodology is proof that these workers do not understand this class of virus. XMRV has never been found in CSF. They should have been using the methods of Lo/Atler as well as the ones by Lombardi. At least Lo and Atler could detect polytropic MRV RNA in plasma. Lombardi et al did not demonstrate that their assays could do that.
Why on earth did they not look for XMRV using proven serology methods which are much more sensitive than PCR namely IHC or Fish? They would have needed to take the required volume of CSF though, around 10 ml not 100 microlitres…
But then, perhaps they didn’t want to find anything, or perhaps they didn’t have the funding to do the job properly.
Thanks for all the help to improve my understanding.
I was a high-flying private banker (steady!) before this all hit – and you know how dense we all are.
Still I am finding it quite fascinating – if completely over my head.
If my niavity offends it isn’t ever meant – I just get fed up with some of the other sources for this information.
Very conspiratorial a lot of it and quite divisive.
So thanks 🙂
There can be no doubt that certain groups are ignoring the facts and the scientific method. That’s not a theory though.