Addenda to the Science paper

[V99]

Don't think this has been posted?

http://www.landesbioscience.com/journals/40/article/12486/

Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome
Judy A. Mikovits, Vincent C. Lombardi, Max A. Pfost, Kathryn S. Hagen and Francis W. Ruscetti

Volume 1, Issue 5
September/October 2010

A PDF is not available for this article

In October 2009, we reported the first direct isolation of infectious xenotropic murine leukemia virus-related virus (XMRV). In that study, we used a combination of biological amplification and molecular enhancement techniques to detect XMRV in more than 75% of 101 patients with chronic fatigue syndrome (CFS). Since our report, controversy arose after the publication of several studies that failed to detect XMRV infection in their CFS patient populations. In this addenda, we further detail the multiple detection methods we used in order to observe XMRV infection in our CFS cohort. Our results indicate that PCR from DNA of unstimulated peripheral blood mononuclear cells is the least sensitive method for detection of XMRV in subjects' blood. We advocate the use of more than one type of assay in order to determine the frequency of XMRV infection in patient cohorts in future studies of the relevance of XMRV to human disease.


Authors

Judy A. Mikovits
Whittemore Peterson Institute; Reno, Nevada USA
Vincent C. Lombardi
Whittemore Peterson Institute; Reno, Nevada USA
Max A. Pfost
Whittemore Peterson Institute; Reno, Nevada USA
Kathryn S. Hagen
Whittemore Peterson Institute; Reno, Nevada USA
Francis W. Ruscetti
Laboratory of Experimental Immunology, Cancer and Inflammation Program; National Cancer Institute-Frederick; Frederick, MD USA

 

[Megan]

Sam Carter just posted on the other thread that the full text of this one is now out.

http://www.landesbioscience.com/jour...tisVIRU1-5.pdf

or if that doesn't work

http://www.landesbioscience.com/journals/40/article/12486/
then download pdf.

Very interesting new information on the original Science article. Explains some of the discrepant results between the studies.

 

[dannybex]

I posted this on the other thread...but am puzzled:

How did the 67% reported in the original paper turn into 75% in this addendum?

I don't get it...but my brain isn't working and am certainly no scientist.

???

"In that study, we used a combination of biological amplification and molecular enhancement techniques to detect XMRV in more than 75% of 101 patients with chronic fatigue syndrome (CFS)."

 

[V99]

They say they used a combination of

biological amplification and molecular enhancement techniques to detect XMRV in more than 75% of 101 patients

and that

we showed that DNA of 7 of 11 patients exhibited the expected gag and env PCR amplification products from single-round PCR with XMRV primers. We included this figure to demonstrate that nested PCR, which inevitably raises questions of contamination, is not essential to detect XMRV in highly viremic ME/ CFS patients. The remaining 90 samples described in the paper exhibited very few XMRV-gag specific PCR products and no env specific PCR products following single round DNA PCR of DNA of unstimulated PBMCs. In contrast, when cDNA was prepared from PBMCs, 67% of thesamples exhibited gag products upon nested PCR, though PCR with nested env primers did not result in detectable products from these samples

 

[V99]

This is from the science paper:

Studying peripheral blood mononuclear cells (PBMCs) from CFS patients, we identified DNA from a human gammaretrovirus, xenotropic murine leukemia virus–related virus (XMRV), in 68 of 101 patients (67%) as compared to 8 of 218 (3.7%) healthy controls.

 

[ixchelkali]

NCI used CDC's Assay!

After we developed a sensitive cell culture assay for detection of XMRV, we assayed our cell lines and patient material with a highly sensitive assay (developed and kindly provided by Bill Switzer, CDC).

So maybe that's why Bill Switzer is giving the presentation on developing XMRV assays at the XMRV conference in September.

 

[SOC]

<<After we developed a sensitive cell culture assay for detection of XMRV, we assayed our cell lines and patient material with a highly sensitive assay (developed and kindly provided by Bill Switzer, CDC). >>

So maybe that's why Bill Switzer is giving the presentation on developing XMRV assays at the XMRV conference in September.

And yet Switzer didn't find XMRV in any samples in the latest Retrovirology paper? Not even a background amount in the control group? Was the sample size too small to find 2-4% infection in the general population?

NCI? This is Ruscetti we're talking about -- who used Switzer's assay?

 

[usedtobeperkytina]

See Susan Vernon's response.

Tina

 

[firefly]

Anyone a Harry Potter fan? It's starting to look like the loyalties, alliances, institutional maneuverings, CYA countermeasures ecc. are all going to turn out to be far more complex than a linear CDC versus NCI/FDA/WPI confrontation. Perhaps Switzer=Snape. That could be kind of fun.

 

[cfssufferer]

I may have overlooked something but I think they only used Switzer's assay to rule out contamination. All of the cell lines and 101 patient materials tested negative for mouse contamination.

 

[SOC]

I may have overlooked something but I think they only used Switzer's assay to rule out contamination. All of the cell lines and 101 patient materials tested negative for mouse contamination.

Ah, thank you. That probably explains it.

 

[George]

Anyone a Harry Potter fan? It's starting to look like the loyalties, alliances, institutional maneuverings, CYA countermeasures ecc. are all going to turn out to be far more complex than a linear CDC versus NCI/FDA/WPI confrontation. Perhaps Switzer=Snape. That could be kind of fun.

(big grins) yeah Snape turned out to be a major player and more or less a good guy. But man I love to hate him!

highly viremic ME/ CFS patients.
and here
required us to
use samples from a subset of chronically ill
patients we had observed to have persistent
viremia.

I noticed this statement. Does anybody see anything or know of anything about "Highly Viremic" aspect of this?

also

Submitted: 02/26/10
Revised: 05/20/10
Accepted: 05/24/10

But not released for pre publish till July 4th and not e-published till July 29th and it looks like this is for the Sept/October print edition. Man, I hope PNAS doesn't do this with the Alter Paper.

 

[ixchelkali]

I may have overlooked something but I think they only used Switzer's assay to rule out contamination. All of the cell lines and 101 patient materials tested negative for mouse contamination.

That's right. And somehow I don't find that reassuring. Um, "We know we didn't have contamination, because we checked for it using an assay from the guys who can't find XMRV even when it's there." What's wrong with this picture?

I'm sure the science gurus would reassure us that it's much more complex than that and we shouldn't worry our pretty little heads.

 

[Megan]

See Susan Vernon's response.

Tina

Tina, what response?

 

[Megan]

That's right. And somehow I don't find that reassuring. Um, "We know we didn't have contamination, because we checked for it using an assay from the guys who can't find XMRV even when it's there." What's wrong with this picture?

Judy talked about using this assay in her webinar in January, so that was a long time ago, and this paper has obviously been written a while back, so both would have happenned well before the release of the CDC results.

 

[George]

A nice Summary of the problems from the other papers

publication, two papers from the United
Kingdom4,5 and a paper from the
Netherlands6 have appeared in which
the authors report the lack of detection
of XMRV PCR products from DNA of
unstimulated PBMCs, using patient populations
selected by only the Fukuda criteria
or the Oxford criteria rather than both
Fukuda and CCC criteria. We regret that
these authors did not request positive control
samples of our patients who exhibit
XMRV PCR products even when assayed
by the least sensitive detection method,
namely PCR of DNA from unstimulated
PBMCs. Given that only 7% of our 101
patients PBMCs exhibit products upon
DNA PCR (Table 3 and 4), and that a
number of patients were included in the
UK studies who do not fulfill the CCC
criteria, very few, if any, of the samples
would be expected to be positive by DNA
PCR. We also note that both studies followed
different methods than ours for
blood collection, DNA quantities and
isolation and PCR, possible sources of
the disparate results. The XMRV detection
results of the 101 patients are listed
in Table 4.

This paper was written in Feb of 10 and revised for publishing in May. When was the CDC paper written does someone have the date handy?

 

[Hope123]

Tina is correct. I have somewhere in my notes that Dr. Mikovits mentioned that "Bill Switzer" helped eliminate the idea of contamination in their studies. If I understand right, it's not just about finding XMRV but finding subtypes of MLVs commonly known to be contaminants in labs.

 

[George]

Tina is correct. I have somewhere in my notes that Dr. Mikovits mentioned that "Bill Switzer" helped eliminate the idea of contamination in their studies. If I understand right, it's not just about finding XMRV but finding subtypes of MLVs commonly known to be contaminants in labs.

Exactly! So this quote

After we developed a sensitive
cell culture assay for detection of XMRV,
we assayed our cell lines and patient material
with a highly sensitive assay (developed
and kindly provided by Bill Switzer, CDC)

Means that Switzer et al turned around and stabbed everybody in the back when they published the CDC study. It was not only Dr. Alter and Dr. Lo who were "surprised" by both the results of the paper as well as the "lie to your face" factor here. WTF mates????

 

[acer2000]

I read through the PDF posted earlier in the thread and came away with both more knowledge about how they did the experiments in the Oct 2009 Science study and also with more questions.

1) It was nice that they listed all 101 patients in a table along with the testing methodologies they used and the results. But why didn't they test all of the samples using all of the methodologies? That doesn't make any sense to me. Maybe they tested them in a stepwise fashion, using the least sensitive/easiest assays first, and only moving on to the more sensitive ones if the samples were negative from the first tests. Is this the case?

2) Also, is there a similar table for the controls? Did they test all of the controls using all of the methods? Did they test them in a stepwise fashion, using the least sensitive methods first, and then only proceeding to the others if the samples were negative? ie. did they just do PCR on the controls, or did they do cultures and "stimulate" the cells before PCR as they did with the non-controls? I am assuming they did, since to be a real "controlled" study you have to use the same methodology on both the cases and controls. Anyone know more details?

3) Finally, although they tested the prostate cancer cell lines for evidence of mouse cell contamination:

After we developed a sensitive cell culture assay for detection of XMRV, we assayed our cell lines and patient mate- rial with a highly sensitive assay (developed and kindly provided by Bill Switzer, CDC) to detect the presence of mouse tissue con- tamination by the identification of murine mitochrondial cytochrome oxidase by real time PCR. All of the cell lines and 101 patient materials tested negative for mouse contamination.

Did they assay the LNCaP cell lines for MLV or XMRV contamination (not just mouse cells) before they used them in experiments? Is it even possible that MLV could be growing in a cell line independent of mouse tissue? Is it possible that those lines were not contaminated with mouse cells or MLV, but may have had human XMRV already growing in them "from the factory"? (They are *prostate cancer* lines - a cell type supposedly associated with XMRV). It seems that every culture would be positive in that case, even in the controls, but since we don't know what tests they did on the controls, its hard to say. And there was a single sample in that table that was cDNA nested PCR positive but not positive by culture. Which would re-affirm that the cell line didn't already have XMRV in it before the experiment. But then you'd think if the blood was positive via PCR, for sure it would culture positive? Kind of confusing.

[EDIT: Maybe line 7 in Table 2 shows the LNCaP cell line testing negative via PCR, indicating that the LNCaP line itself was free of human XMRV before it was used in culture experiments? Is that what this means?]

Perhaps some others can take a look at this addendum and let me know if they addressed this stuff. Its entirely possible that I am just misreading it. But those were the questions I came up with.


Thoughts?

 

[V99]

I believe they said they could not do every test because of time constraints and money.