Negative study of CFSers cerebrospinal fluid for XMRV and other viruses

[Esther12]

Isn't Singh working on a spinal fluid study? Wonder how that's going.

http://onlinelibrary.wiley.com/doi/...+on+Saturday,+5th+Feb+between+10:00-12:00+GMT

Only a small abstract:

Analysis of cerebrospinal fluid from chronic fatigue patients for multiple human ubiquitous viruses and XMRV

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. Ann Neurol 2011.

 

[Esther12]

Author info:

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
2. 2
Ibis Biosciences, Inc. Carlsbad, California, USA
3. 3
Albert Einstein School of Medicine, Bronx NY and Beth Israel Medical Center, Department of Pain Medicine and Palliative Care, New York, NY 10003

 

[August59]

I believe there is another study by Br. Barinuk that also looks at CSF. I don't know for sure if it looked for XMRV, but I do know Cort participated in part of the study.

 

[Esther12]

This could be the announcement to the study... shame it came up with nothing interesting:


Infectious Triggers In Chronic Fatigue Syndrome
Steven E. Schutzer, MD
University of Medicine and Dentistry of New Jersey – New Jersey Medical School

Funding Institute: National Institute of Allergy and Infectious Diseases
Project Start Date: March 1, 2010
Project End Date: February 29, 2012
Funding for 2010: $273,000

DESCRIPTION (provided by applicant): chronic fatigue syndrome (CFS) is a major disabling illness of unknown etiology. It directly impacts more than four million Americans. Infectious agents have been highly suspected but none have ever been validated. Obstacles to their discovery have been lack of a broad approach that can both detect the presence of multiple microbes in a single sample and detect novel or variants of microbes. This has been further hampered because more than 99% of all microbes cannot be cultured and may go undetected. In contrast to past limitations, we will use a new approach that can surmount these obstacles. The discovery of potential pathogens in cerebrospinal fluid of CFS would be field-altering in terms of approach to the study of the disease and possible early detection, prevention and treatment. This could be the gateway step to generate new hypotheses and begin investigation into a microbe's causal association and ways to prevent (egg vaccine) or counteract the effects of a microbial pathogen.

PUBLIC HEALTH RELEVANCE: chronic fatigue syndrome is the major acquired disease of productive adults. The cause remains unknown. If we can uncover an infectious cause, which is suspected, diagnostics and therapies may be developed to decrease costs and suffering to the individual and the burden on our health care system and economy.

 

[August59]

Quick observations:

They keep using simply PCR techniques which we already know doesn't work and probably will not work until they get the proper assays developed. This project started back in March 2010 and was not scheduled to end until February 2012. I imagine CSF will have to be preped and handled very specifically just as blood is.

What cohort did they try to use?

This study was funded by the NIAID group and the grant was awarded to whom???
The University of Medicine and Denistry
Ibis Biosciences
Dept. of Pain Medicine and Pallative Care

How did they get a grant? I geuss you have to be a dentist to research XMRV

 

[Cort]

What a shame that Natelson couldn't find it. The methodology will always be a question with XMRV until they come to a consensus on an assay. Unless they are trying something novel I wonder if everyone should just wait..

That is a weird grant designation I grant )) but I believe that the medical University is Uni of Medicine and Dentistry for some reason. He used to do alot of work on CFS (ran one of the NIH Research Centers) and that's where he's always worked out of.

 

[Cort]

Baraniuk was looking for pathogens in CSF samples. When I saw him about halfway through his study he said he hadn't found much. I see this group didn't find viruses either. I don't know if he started looking for XMRV. He did do a review article on XMRV - which did indicate some interest - so he may have picked it up later.

 

[Dolphin]

Some of the testing was by VipDX. Unfortunately it also was negative.

Co-cultivation of Material from CSF Samples with Permissive Reporter Cell Line
We used VipDx in the capacity of a service laboratory. The VipDX XMRV PCR/culture test is a 2 stage process. First clinical material (cells or fluid) are probed by PCR directly for XMRV2. Secondly and independently the clinical sample is co-cultivated with a cell line(LNCaP) permissive for XMRV infection. After weeks of culture or visible transformation, the exposed cell line is probed by PCR for XMRV. The same cell line exposed to known XMRV negative human material serves as a negative control to be assayed in the same fashion. The same permissive LNCaP cell line, not exposed to any potential XMRV human cells or fluid, can also serve as a negative control. Positive controls are their in-house native controls, previously found to be XMRV positive. To maintain the integrity of a mutually agreed upon blinded process, our samples were submitted to the VIPDx laboratory for full processing in their assay.

Results:

Blinded Independent Evaluation of Samples for XMRV by PCR
Nucleic acid extracts from 43 CFS patients which were pooled into two groups and positive controls (distilled water and XMRV cDNA), and negative controls (distilled water) were all negative except for the spiked control sample, containing cDNA from XMRV.

Blinded Independent Evaluation of extracts for XMRV by PCR after Co-cultivation
Co-cultivation experiments also yielded negative results.

(I probably won't post any more on this thread - I'm out of my depth in such discussions and the threads usually gets very long).

 

[Cort]

Thanks Dolphin, I guess we can assume that it's not in the spinal fluid???

 

[Cort]

The discovery of potential pathogens in cerebrospinal fluid of CFS would be field-altering in terms of approach to the study of the disease and possible early detection, prevention and treatment. This could be the gateway step to generate new hypotheses and begin investigation into a microbe's causal association and ways to prevent (egg vaccine) or counteract the effects of a microbial pathogen.
.

Nice catch Esther! What shame it didn't work out....that was quite a study. I didn't even know it was underway...

 

[Bob]

Thanks Dolphin, I guess we can assume that it's not in the spinal fluid???

I haven't looked at the methodology of this study, but remember that Judy Mikovits says the virus can be destroyed before it's tested for, if it's frozen and thawed and refrozen etc. So there could be sample collection, processing and storage issues that are causing the virus not to be detected in this study, even if they used VipDX to do the testing. So maybe it's too early to rule it out being in the spinal fluid, if the methodology isn't perfect.

 

[acer2000]

Does anyone know if MLV type viruses are typically found in CSF in other mammals? It was a good idea to look there, but I'm curious... Are other retroviruses that effect the CNS present in the CSF? Like HTLV, or HIV? [Edit they claim in the paper that HIV can be found in CSF]

It does appear on the surface at least... that in addition to their in house experiements they actually sent all of their samples to VIPdx to be tested via the culture + PCR test that they currently perform.

I suppose that still leaves open the question of if XMRV is even in CSF (was it in the monkeys?) or if there was some part of processing the CSF that caused a change in the result.

I'd be curious about Dr. Mikovits's opinion on this as well obviously.

 

[Enid]

Thanks for interesting thread - must say I was pretty convinced something should have be found at my lumbar puncture stage. And more especially since pressure was high which Consultant Neurologist couldn't explain. At that stage sleeping mostly barely able to speak or walk. Hope this research will go on.

 

[jace]

Of course 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 - it means they used their protocols, not 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.

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!

If someone had missed adenovirus in the CSF using this procedure they would have been sued for millions of dollars and struck off the register

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

 

[omerbasket]

Do anyone here know if other viruses, such as EBV, CMV, parvovirus B19, enteroviruses etc. were found in previous studies in the cerebrospinal fluid of CFS patients? Or even in healthy controls?
Because it seems quite odd to me that they didn't just not-find any XMRV in the CSF, but they didn't find any other viruses there, although they have tested for many of them:

All 43 individual CSF samples (based on available volumes) from CFS patients analyzed with
the broad range RT-PCR/ESI-MS8 were negative for Human Adenoviruses, Alphaviruses,
Herpes viruses (HHV 1, 2, 3, 4, 5, 8 ), Human Parvovirus B19, Dengue viruses 1-4, WNV, JEV,
SLE, Enteroviruses A-D, and Coxsackieviruses.

You should know that HHV-4 is EBV and HHV-5 is CMV.

What I'm thinking is - and I'd be very glad to hear your opinion about that - perhaps their viruses were killed, in their lab and also in the samples shipped to VIP Dx? I mean, can the type of tube, or other things that they did could have killed all of these viruses if they were there?

 

[jace]

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/ency/article/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.

Also 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.

 

[jace]

Some further information has come to light:

They had 43 patients, but they sent VIPDx a total of five vials.

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?

 

[eric_s]

I think the fact that VIP Dx didn't find XMRV here is not even a bad thing. VIP Dx has found XMRV in many cases before. To me, it would be more worrying if VIP Dx or the WPI always finds something where others find nothing.

 

[August59]

But in spite of all of the concerns - They received a $273,000 grant from the NIH (NIAID) and got it published. From start to publication was 10 months!! This should be used as an example on why a grant should have never been approved much less make it to publication.

With McClure being on the panel now I'll imagine that there will be plenty of these bogus studies. Bogus in execution as I think the CSF should still be looked at very hard, but studies like this will only make it harder.

 

[Esther12]

From what people have posted so far, this seems like a decent study. They might have missed someting, or there might just not be virus to be found in CSF, but to me, this looks like the sort of research I'd like to see. It looks like they weren't initally focusing upon XMRV, but trying to find other infections instead, so maybe the XMRV part wasn't as ideal as we'd have liked, but I've not seen anything posted that would make me think it was bogus, or intentionally flawed.