XMRV CFS UK study #II

[_Kim_]

I moved our new UK XMRV+ member's posts over to their own thread:

Welcome wideawake - one of the UK 14 (XRMV+)

 

[flex]

Today I did a scientific experiment on an apple. I looked at it. It looked like an apple. I smelt it, it smelt like an apple. I tasted it, it tasted like an apple. I had a team of scientists examine it and they all agreed, it was indeed an apple. This was a conclusive result and the science was watertight.


A team of scientists in Nevada followed the following criteria for a scientific experiment and discovered that XMRV a retrovirus is heavily implicated in the Neurological disease ME:

Ask a Question
Do Background Research
Construct a Hypothesis
Test Your Hypothesis by Doing an Experiment
Analyze Your Data and Draw a Conclusion
Communicate Your Results

Their results were supported by two other major organisations, the CC and the NCI. After six months peer review the study was published in the worlds leading magazine.


Consider the two examples above. Both scientific, both with conclusions. To those saying that they find no flaws in the latest UK study and the science was watertight consider the purpose and the usefulness of what was measured.

 

[ukxmrv]

SGUL (DR Kerr) contributed 142 CFS patients for this study. They may have been earlier blood samples taken from his gene expression work. Just a guess as the locations mentioned in the new paper are similar to the locations he got patients from for earlier papers (with the exception of New York which he didn't use here)

Here is extracts from some of his gene expression papers on patient selection

Subject enrollment, clinical characterization, and blood sampling
----------------------------------------------------------------------------

Twenty-five patients with CFS/ME from the Dorset CFS Service in southeast England were enrolled for the microarray study. Patients with CFS/ME whose blood was used for subsequent PCR studies comprised those in the microarray study along with an additional 30 patients from clinics in 3 United Kingdom cities (Dorset, Bristol, and London; 1 patient from Leicester was under the care of a clinic in London) and New York, New York.

CFS/ME was diagnosed on the basis of Centers for Disease Control and Prevention (CDC) criteria [1].

Patients with psychiatric disease were excluded on the basis of findings of the Minnesota International Neuropsychiatric Interview, thus ensuring that no patients had major psychiatric disease or were abusing alcohol or drugs.

In addition, patients who had smoked tobacco during the previous 12-month period and/or had taken antibiotics, steroids, or antidepressants during the previous 3-month period were excluded from the study.

For all enrolled subjects, according to the recommendations of the International CFS Study Group,7
severity of physical and mental fatigue was assessed using the Chalder Fatigue Scale;8
level of disability was assessed using the Medical Outcomes Survey Short Form-36 (SF-36);
accompanying symptoms were characterised using the Somatic and Psychological Health Report (SPHERE);
sleep abnormalities were assessed using the Pittsburgh Sleep Questionnaire;
and assessment of type and severity of pain was performed using the McGill Pain Questionnaire.

For the CFS/ME patients, neurocognitive testing was performed using the Spatial Span (SSP) and Verbal Recognition Memory (VRM) modules of the Cantab software (Cambridge Cognition, UK), which showed abnormal results in CFS/ME.6,9

 

[Gerwyn]

SGUL (DR Kerr) contributed 142 CFS patients for this study. They may have been earlier blood samples taken from his gene expression work. Just a guess as the locations mentioned in the new paper are similar to the locations he got patients from for earlier papers (with the exception of New York which he didn't use here)

Here is an extract from one of his gene expression papers on patient selection

Subject enrollment, clinical characterization, and blood sampling
----------------------------------------------------------------------------

Twenty-five patients with CFS/ME from the Dorset CFS Service in southeast England were enrolled for the microarray study. Patients with CFS/ME whose blood was used for subsequent PCR studies comprised those in the microarray study along with an additional 30 patients from clinics in 3 United Kingdom cities (Dorset, Bristol, and London; 1 patient from Leicester was under the care of a clinic in London) and New York, New York.

CFS/ME was diagnosed on the basis of Centers for Disease Control and Prevention (CDC) criteria [1].

Patients with psychiatric disease were excluded on the basis of findings of the Minnesota International Neuropsychiatric Interview, thus ensuring that no patients had major psychiatric disease or were abusing alcohol or drugs.

In addition, patients who had smoked tobacco during the previous 12-month period and/or had taken antibiotics, steroids, or antidepressants during the previous 3-month period were excluded from the study.

that is very interesting psychiatric disorders dont of course include depression do we know any more about the patients supplied for this study does he look for post exertional malaise

 

[starryeyes]

I had to reformat ladybugmandy's post so I could understand it so I figured I would repost it here in the reformatted version for all of you:

More information regarding XMRV studies:

1. The authors of the Science paper established the existence of XMRV as an infectious human blood borne retrovirus for the first time in blood of patients diagnosed with CFS. Previous studies had established the presence of XMRV sequences and protein in human prostate tissue. The basic premise that XMRV may be capable of causing disease has not been questioned.

2. In the Science paper, the presence of XMRV in well-characterized patients with CFS was established using multiple technologies:

a) PCR on nucleic acids from un-stimulated and stimulated white blood cells;
b) XMRV protein expression from stimulated white blood cells;
c) virus isolation on the LNCaP cell line; and
d) specific antibody response to XMRV.

3. The authors of the two UK studies did not attempt to "replicate" the WPI study. Replication requires that the same technologies be employed.

4. The collection, preparation and storage of DNA were completely different between the Science and UK papers. The latter studies do not show data on how blood was harvested and stored. Nor do the studies disclose the quantity of isolated cells. Insufficient number of cells analyzed may result in failure to detect a low copy virus like XMRV, regardless of the sensitivity of the assay. Neither UK study provides details to allow interpretation of how many white blood cells were analyzed.

5. Patient selection was based on different definitions of CFS.

6. The UK authors were unable to detect XMRV, even though 4% of healthy individuals were found to be infected in the US. Japanese scientists detected XMRV in 1.7% in healthy blood donors in Japan. The two previously identified human retroviruses have distinct geographical
distribution.

7. Perhaps the most important issue to focus on is the low level of XMRV in the blood. XMRV is present in such a small percentage of white blood cells that it is highly unlikely that either UK study’s PCR method could detect it using the methods described. Careful reading of the Science paper shows that increasing the amount of the virus by growing the white blood cells is usually required rather than using white blood cells directly purified from the body.

When using PCR alone, the Science authors found that four samples needed to be taken at different times from the same patient in order for XMRV to be detected by PCR in freshly isolated white blood cells. More importantly, detection methods other than PCR showed that patients whose blood lacks sufficient amount of XMRV detectable by PCR are actually infected. This was proven by the isolation of viral proteins and the finding of infectious XMRV isolated from the indicator cell line LNCaP. The authors of the Retrovirology paper admit that their neutralization assay did not detect bacterially expressed XMRV gag and that positive control sera was needed to validate their assay.

The WPI’s monoclonal antibodies specifically and sensitively completed the immune response demonstrating the assays' sensitivity and specificity for XMRV envelope. Neither UK study requested positive control blood, plasma or nucleic acids from the WPI.

Simply stated the only validated reliable methods for detecting XMRV in CFS patients, to date, are the methods described in Science. Failure to use these methods and validated reagents has resulted in the failure to detect XMRV. A failure to detect XMRV is not the same as absence of XMRV.

Thank you so much for posting this ladybug!

 

[ukxmrv]

Gerwyn,

The Fukuda criteria was used but one table looks at symptoms experienced by the subjects. It's split into 2 parts

Table 1. Demographic and clinical characteristics and results of diagnostic tests for patients with chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) and healthy blood donors involved in microarray and real-time polymerase chain reaction (PCR) studies

Postexertional Malaise

23/25 (microarray patients) where 25 = total patients for that group

and

47/55 (PCR) and 55 equals total patients for that group

 

[Adam]

It's bloody fantastic. It's about time one of the UK advocates had the bollocks to speak up. <---- practicing my newly learned UK slang

Bollocks is arguably the most amusing of our vast array of slang/swear words here in the UK. There is another expression, Kim, which also encompasses this colourful word, which you may not yet be acquainted with, which is... the Dog's Bollocks (don't fret George no dog's testicles were harmed in the making of this post).

It could describe Judy Mikovits (i'm sure she'd be proud) or Malcolm Hooper or Dan Peterson or anyone one of our growing list of heroes.

It simply means the very best.

Phoenix Rising IMHO is the Dog's Bollocks.

 

[julius]

flex,
your apple experiment didn't really follow those steps. The main one is that background research would have shown you that there are certain defining characteristics of an apple. Probably genetics are the best tool to use today. But the smell, taste etc. aren't how you identify an apple. Also, what examinations did your team of scientists do?

As far as the UK studies... watertight, I definitely didn't say that. And usefulness, I agree, not particularly useful. But still science by definition. Science can be done well, and it can be done poorly. WPI did it exceedingly well, and the UK teams did it somewhat poorly. Well below the standard set by the WPI.

I think the big problem is bias. I think Gerwyn's post about the hypothetico deductive model was largely trying to address that. That model is a description of the classic scientific method which tries to address some of the problems inherent in the classic model...bias being one of them.

If, for example, the suspicions of some of us (definitely including myself) that the IC study was biased are true, then it would not be science. Even by the classic model. The act of setting a goal (ie; to not find XMRV) and designing a 'study' to achieve that goal is not science. A hypothesis is not a goal, it is an educated guess. But for now we have no 'proof' that is what occurred, so we have to (unfortunately) treat it as science.

As far as the conclusions, I was wrong. I said the conclusions of the two studies were that those methods can't find it. But really the only conclusions are that they didn't find it. I made an assumption it was there based on the fact that we know that it is in the UK from the commercial testing.

BTW Gerwyn, I looked for the statement about replication in the IC study and didn't see it.

 

[fingers2022]

Professor Hooper is on our side big-time...
He's been a long-time advocate for the biomedical science of ME.

"Professor Malcolm Hooper will be the chairman for the 5th Invest in ME International ME/CFS Conference 2010"
The Invest in ME conference is always really interesting, and very scientific, and Judy Mikovits will be speaking at this year's conference.
Here's the website link for the Invest in ME London conference in case anyone is interested:
http://www.investinme.org/IIME Conference 2010/IiME 2010 International ME Conference Home.htm

Hi Bob

Thanks for the link, but 5 years and not much to show. We need to get to the bottom of the XMRV stuff asap, then we can all crawl back into our holes and manage our suffering as best as we can.....or move on with renewed hope.

It's the asap that I'm interested in - it needs managing, and scientists can't do that I'm afraid.

 

[_Kim_]

I had to reformat ladybugmandy's post so I could understand it so I figured I would post it here in the reformatted version for all of you:

teejkay, ladybugmandy...

Maybe I've missed this but WHO is the source of this information??

 

[Cort]

As I remember it was reported it was from an email from Dr. Mikovits. It certainly sounds like it provides several very good reasons why those studies might fail to find XMRV. I HOPE it was from Dr. Mikovits - it seems like a very strong statement; I guess we'll see when the WPI gives their response. (When are they going to respond?)

(I heard from someone that the CDC is digging really deeply into this virus - they're looking at their samples and other groups samples - and that they are finding something. I don't know what it is - it could be a endogenous retrovirus - or it could be 'XMRV' or whatever. It was funny because the news really excited me and then I immediately turned to the forums - and there was the UK study! Blah! Anyway the rumor suggested that this isn't going to be like the DeFreitas experience;that these teams are going to try hard to get to the bottom of this.

By the way these studies must be costing oodles of money. I guess we'll never know but, taking a wild guess, I imagine all told they've spent more in the last couple of months than they have for several years. )

Thanks for that great formatting job Teejkay!

 

[_Kim_]

Bollocks is arguably the most amusing of our vast array of slang/swear words here in the UK. There is another expression, Kim, which also encompasses this colourful word, which you may not yet be acquainted with, which is... the Dog's Bollocks (don't fret George no dog's testicles were harmed in the making of this post).

It could describe Judy Mikovits (i'm sure she'd be proud) or Malcolm Hooper or Dan Peterson or anyone one of our growing list of heroes.

It simply means the very best.

Phoenix Rising IMHO is the Dog's Bollocks.

Thanks Adam . Katie tried to explain this to us in chat, but I was still confused

It gets even more confusing if I were to say that George is the Dog's Bollocks :Retro tongue:

 

[_Kim_]

As I remember it was reported it was from an email from Dr. Mikovits. It certainly sounds like it provides several very good reasons why those studies might fail to find XMRV. I HOPE it was from Dr. Mikovits - it seems like a very strong statement; I guess we'll see when the WPI gives their response. (When are they going to respond?)

Thanks for that great formatting job Teejkay!

Is this the same email that was posted on this thread earlier and then removed because it was believed to be fraudulent?

 

[Hope123]

There has been a lot of discussion here about 'science', some of it has been a bit misinformed. Something is 'science' if and only if it follows the scientific method. The scientific method is very straightforward. Although there are some slight variations, this is basically what it is;


Ask a Question
Do Background Research
Construct a Hypothesis
Test Your Hypothesis by Doing an Experiment
Analyze Your Data and Draw a Conclusion
Communicate Your Results

That's it. If it follows this formula it is science, of not it is not.

Replication vs. Validation

This has also come up a lot.

Neither of the UK studies were a replication study.
Neither of the UK studies claimed to be a replication study.
Neither of the UK studies were under any obligation to be a replication study.

But they did follow the scientific method.
However, the most recent one did make a claim in it's title which was not supported by the data. They reported the absence of XMRV in CFS. The data only suggests that they did not detect it.


Personally I think the WPI paper leads to two lines of investigation;
1) what is the nature of XMRV and how can we find it/study it (ok, that's kind of sloppy)
2) What is the relationship between XMRV and CFS

The first line should involve all kinds of methods. Sticking to only one method here won't move our understanding of the virus forward in any great way.
The second question should stick to the methods which have been shown to find it.

The two UK studies were trying to address the second question. But, by using such different methods, only addressed the first question. The real conclusions of those two studies are
1) you can't find it this way
2) you can't find it that way

I agree with Julius. Here's the process in a more detailed flow chart. It's aimed at young students but the process is the same at higher levels.

http://www.sciencebuddies.org/science-fair-projects/project_scientific_method.shtml

An important piece is the arrow with "Think! Try again." To progress, scientists need to take their conclusions, whether it confirms or doesn't confirm their hypotheses and continue to generate new questions/ hypotheses. The part where the hypothesis is 'false' or 'partially true' leading to communication of results is a major issue in CFS research.

Some CFS clinicians, to remain unamed, make all sorts of claims about treatments for CFS but never publish anything - not even a single case report where you write only about one patient. Others, because of the all mighty dollar, also never publish anything (I'm looking at you Ampligen) because of business concerns.

NEGATIVE results are just as important as POSITIVE results. We can debate the pros and cons of studies when they are published but not if we never hear/read about them. Well-designed studies with NEGATIVE results help scientists to narrow down possibilities, concentrate their efforts in other hypotheses,etc.

 

[julius]

Is this the same email that was posted on this thread earlier and then removed because it was believed to be fraudulent?

No. That one was just a bit of a rant.

 

[Gerwyn]

Gerwyn,

The Fukuda criteria was used but one table looks at symptoms experienced by the subjects. It's split into 2 parts

Table 1. Demographic and clinical characteristics and results of diagnostic tests for patients with chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) and healthy blood donors involved in microarray and real-time polymerase chain reaction (PCR) studies

Postexertional Malaise

23/25 (microarray patients) where 25 = total patients for that group

and

47/55 (PCR) and 55 equals total patients for that group

Thank you very much

 

[julius]

hope123,

That's funny, if you look at my outline of the scientific method, you'll see that I copied and pasted it from the same page you linked.

I chose it because it was simple and gave a basic idea of the theory.

 

[Advocate]

Someone commented that Judy Mikovits's body language in the webinar showed confidence. So I'm hoping that she knows some good things she can't talk about yet. I've bolded one sentence below that suggests she has decided to be careful.

I've lost track of where I got these comments. If they have been posted before, tell me and I'll delete them.

Date: Mon, 15 Feb 2010 10:46:42 -0600
From: Tate Mitchell <tatemitchell@GMAIL.COM>

Professional comments re: Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue Syndrome

In a recent blog posting discussing the pros and cons of the two currently
published studies on XMRV and CFS, which itself is one of the better reviews
I've read, the author refers to a post in a discussion on Faculty of 1000,
which appears to be a kind of forum for professionals to comment on research
papers. There are several comments made, including one dissenting opinion
and an author's response by Dr. Mikovits.

Author Response:
Judy Mikovits, Whittemore Peterson Institute, Reno, United States

This dissent first discusses "the cause of CFS".

We did not imply that XMRV caused CFS. We specifically state that our
observation "raises several important questions".

Is XMRV infection a causal factor in the pathogenesis of CFS or a passenger
virus in the immunosuppressed CFS patient?

The work presents a testable hypothesis that XMRV has a role in CFS
pathogenesis.

The key task for the scientific community is to define the scope of human
disease associated with XMRV infection.

We were highly concerned and vigilant about taking precautions to avoid PCR
contamination.

This was minimized using laboratory controls such as dedicated first- and
second-round PCR areas in different buildings and consistent treatment of
instruments and work areas with DNA ZAP and UV rays.

Although the original screen was performed using nested PCR, PCR positivity
on a subset of samples was confirmed using single-round PCR in a different
facility (Fig1A).

Although not in the manuscript, PCR status was verified on identical samples
not processed at WPI in a XMRV-free lab at NCI. Moreover, in normal samples analyzed in parallel, amplified sequences were found in only 3.7%, making it unlikely that 67% positivity reflects "uncontrolled PCR contamination".

We should not have discussed unpublished data, particularly considering the lay media.

To discuss more details about our more recent studies would repeat that
error, but we were able to culture XMRV virus from plasma and detect
antibodies in some samples which were PCR-negative (e.g. see #1118 in Fig1,
2A, 2D).

These examples compelled us to further study PCR-negative patient samples.

Flow cytometry data in the study were not meant to determine in vivo levels
of XMRV protein expression or the level of clinical viremia.

The goal was to determine if the XMRV DNA sequences detected represented the presence of infectious viral particles.

The studies of XMRV protein expression in peripheral blood mononuclear cells
(PBMC) were performed after PBMC were activated in culture with
phytohemagglutinin and interleukin-2 for 7-14 days to allow the virus to
spread through the culture. It should have been made clearer.

Although at earlier times these samples did have bimodal peaks, data shown
were when most cells were virus positive.

Not included in the paper was the ability of azidothymidine to block XRMV
spread in vitro. The methodology for the immunoblots was also questioned.

We used monoclonal antibodies which recognized the envelope of all
xenotropic and polytropic but not ectopic murine leukemia viruses and
reacted with XMRV env proteins of expected sizes.

Concerning the relatively weak signal of the positive control, HCD-57/SFFV,
test sample lanes contained 150-200ug of protein, only 30ug was loaded in
the HCD/SFFV lane to prevent the positive control signal from overwhelming
adjacent lanes.

This study was the initial finding of infectious XMRV virions in human blood
and a second association of XMRV and human disease.

Our observation of actively replicating virus, as well as antibodies
directed against XMRV (which were not criticized) in the patient population
examined, strengthened the paper and the hypothesis that this recently
discovered virus is a human pathogen.

Clinically, CFS is a heterogeneous syndrome with diagnosis made on the basis
of the exclusion of other diseases.

Thus, the basis for diagnosis varies greatly and we expect there to be
XMRV-positive and -negative CFS patients. Additional large-scale clinical
studies using control groups are essential to determine whether XMRV is the
cause of CFS.

Also, rigorous independent validation is crucial. To facilitate this, the
NCI, Cleveland Clinic and WPI are making virus reagents available through
the NIH AIDS repository to any academic investigator by contacting the
investigators involved.

Response added 7 Jan 2010

 

[Gerwyn]

I agree with Julius. Here's the process in a more detailed flow chart. It's aimed at young students but the process is the same at higher levels.

http://www.sciencebuddies.org/science-fair-projects/project_scientific_method.shtml

An important piece is the arrow with "Think! Try again." To progress, scientists need to take their conclusions, whether it confirms or doesn't confirm their hypotheses and continue to generate new questions/ hypotheses. The part where the hypothesis is 'false' or 'partially true' leading to communication of results is a major issue in CFS research.

Some CFS clinicians, to remain unamed, make all sorts of claims about treatments for CFS but never publish anything - not even a single case report where you write only about one patient. Others, because of the all mighty dollar, also never publish anything (I'm looking at you Ampligen) because of business concerns.

NEGATIVE results are just as important as POSITIVE results. We can debate the pros and cons of studies when they are published but not if we never hear/read about them. Well-designed studies with NEGATIVE results help scientists to narrow down possibilities, concentrate their efforts in other hypotheses,etc.

Science attempts to explain observable and unobservable phenomena by constucting models and make predictions based on that model aiming to disprove the hypothesis This last bit is critical The flow chart you highlight is positivistic which no longer drives the scientific approach although it is widely held to To adance science the hypothesis and or conclusions must be falsifyable The results/conclusions of these studies are not so no progress

 

[cfs since 1998]

(I heard from someone that the CDC is digging really deeply into this virus - they're looking at their samples and other groups samples - and that they are finding something. I don't know what it is - it could be a endogenous retrovirus - or it could be 'XMRV' or whatever. It was funny because the news really excited me and then I immediately turned to the forums - and there was the UK study! Blah! Anyway the rumor suggested that this isn't going to be like the DeFreitas experience;that these teams are going to try hard to get to the bottom of this.

If that's true, and Reeve's reassignment was related to this, I am wondering why the CDC is having a change of heart. Maybe the CDC has been given a dose of scientific curiosity by all this, or maybe they want to confirm WPI's finding so that they can salvage the CDC's reputation and be seen as the hero?