German Study - No XMRV in CFS & MS

[shannah]

Source: PlosOne
Vol 5, #12 (Preprint)
Date: 24 december 2010
URL:
http://www.plosone.org/article/fetc...leURI=info:doi%2=10.1371/journal.pone.0015632

Edit:
try instead

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015632

No evidence for XMRV in German CFS and MS patients with fatigue
despite the ability of the virus to infect human blood cells in
vitro
---------------------------------------------------------------
Oliver Hohn(1,5), Kristin Strohschein(2), Alexander U. Brandt(3),
Sandra Seeher(1), Sandra Klein(1), Reinhard Kurth(4), Friedemann
Paul(3), Christian Meisel(2), Carmen Scheibenbogen(2,#), Norbert
Bannert(1,5,#,*)
1 Centre for Biological Security 4, Robert Koch-Institute, Berlin,
Germany,
2 Institute for Medical Immunology, Charite - Universitatsmedizin
Berlin, Berlin, Germany,
3 NeuroCure Clinical Research Center (NCRC), Charite Universitats-
medizin Berlin, Berlin, Germany,
4 Robert Koch-Institute, Berlin, Germany,
5 Centre for Retrovirology, Robert Koch-Institute, Berlin, Germany
* E-mail: bannertn@rki.de
# These authors contributed equally to this work.


Received: September 16, 2010; Accepted: November 18, 2010; Published:
December 22, 2010


Abstract

Background
Xenotropic murine leukemia virus-related virus (XMRV), a novel
human retrovirus originally identified in prostate cancer
tissues, has recently been associated with chronic fatigue
syndrome (CFS), a disabling disease of unknown etiology
affecting millions of people worldwide. However, several
subsequent studies failed to detect the virus in patients
suffering from these illnesses or in healthy subjects. Here we
report the results of efforts to detect antibody responses and
viral sequences in samples from a cohort of German CFS and
relapsing remitting multiple sclerosis (MS) patients with
fatigue symptoms.

Methodology
Blood samples were taken from a cohort of 39 patients
fulfilling the Fukuda/CDC criteria (CFS), from 112 patients
with an established MS diagnosis and from 40 healthy donors.
Fatigue severity in MS patients was assessed using the Fatigue
Severity Scale (FSS). Validated Gag- and Env-ELISA assays were
used to screen sera for XMRV antibodies. PHA-activated PBMC
were cultured for seven days in the presence of IL-2 and DNA
isolated from these cultures as well as from co-cultures of
PBMC and highly permissive LNCaP cells was analyzed by nested
PCR for the presence of the XMRV gag gene. In addition, PBMC
cultures were exposed to 22Rv1-derived XMRV to assess
infectivity and virus production.

Conclusion
None of the screened sera from CFS and MS patients or healthy
blood donors tested positive for XMRV specific antibodies and
all PBMC (and PBMC plus LNCaP) cultures remained negative for
XMRV sequences by nested PCR. These results argue against an
association between XMRV infection and CFS and MS in Germany.
However, we could confirm that PBMC cultures from healthy
donors and from CFS patients can be experimentally infected by
XMRV, resulting in the release of low levels of transmittable
virus.

--------
(c) 2010 PlosOne

 

[shannah]

This study came through Co-Cure this morning. I've copied it exactly as reported but the link doesn't appear to be working.

 

[shannah]

Can one of our scientific minds critique this study for us please.

 

[alex3619]

Hi everyone, I haven't read the entire paper yet, but I dismissed this paper as probably not important when I saw it on cocure. First, it was published in PLoS: this is a low rating journal. Second it uses nested PCR, which we already know is problematic. Third, while they did culture for the virus they only cultured for 7 days: we know the WPI finds it takes up to 42 days to culture the virus. On the other hand, they did use Fukuda at least for the CFS diagnoses.

If I get around to reading this paper, which might not be soon, I will post a longer commentary.

Bye,
Alex

ps Almost forget, isn't this another zero zero study? They did however show that XMRV can infect these cultures and is an infective virus. This may be worth a more detailed look.

 

[shannah]

Thanks Alex for taking a look and commenting as quickly as you did.

I knew enough not to be alarmed by it but certainly not enough to understand why not.

In the Nevada Newsmakers clip just before Christmas, I noticed Judy is now saying it can take up to 45 days to culture, up from her previously stated 42.

 

[alex3619]

In the Nevada Newsmakers clip just before Christmas, I noticed Judy is now saying it can take up to 45 days to culture, up from her previously stated 42.

Hi shannah, thanks for this, somehow I missed noticing that detail from Judy - my brain probably heard 42. I wonder if this number is going to keep going up? If extra culturing time increases the capacity to find the virus enough then it may become standard for XMRV testing. Bye, Alex

 

[jeffrez]

So how come they didn't have antibodies?

 

[kurt]

Hi shannah, thanks for this, somehow I missed noticing that detail from Judy - my brain probably heard 42. I wonder if this number is going to keep going up? If extra culturing time increases the capacity to find the virus enough then it may become standard for XMRV testing. Bye, Alex

The original Science article study stated the T and B cells were cultured for 42 days. She can't change that to 45 days now, other labs should be able to find what she found the same way she found it. And even then, they now must run the more robust contamination studies, which WPI should also be doing as well, given the results of the Retrovirology studies. If WPI does not re-run their samples with the more robust contamination studies, they will lose credibility in the scientific community.

 

[alex3619]

If WPI does not re-run their samples with the more robust contamination studies, they will lose credibility in the scientific community.

Hi kurt, I agree, this is a real risk. I think that it will happen (the new tests that is), but it will take a while. I also worry that they need to test their culture medium and other reagents with the alternative mouse DNA tests. Bye, Alex

 

[alex3619]

So how come they didn't have antibodies?

Hi Mr. Kite, there might be a number of reasons. They didn't use XMRV positive human controls to validate their methods. Their antibody methods were from a prior zero zero study, and validated using controls in yet another zero zero study. The goat serum controls were probably from recently infected goats. Why does everyone forget/ignore that the virus disappears from the blood after a number of days? Their antibody tests were to directly find XMRV using XMRV targeted manufactured antibodies, they did not look for natural antibodies to XMRV.

They found when they infected a culture that XMRV was detectable using their PCR test after seven days. Their test does work on tissue that has a heavy viral load.

The good news: reverse transcriptase. They found high RT in the infected culture, but within expected range for controls and CFS patients. In other words the virus is not massively replicating in CFS patients. This is a good thing, but we already kind of knew this from the low copy number issue in CFS patients.

Take home points: their tests should have been expected to not work based on prior studies. They did however show, again, that this is a real virus and not strongly present in the blood, nor massively replicating.

Bye, Alex

 

[Cort]

Second it uses nested PCR, which we already know is problematic.

I've got to look at the paper too but I do believe that nested PCR is what the WPI used in their original study.

I think this is an old study though.....

 

[shannah]

Hi Cort,

This particular study was
Received: September 16, 2010; Accepted: November 18, 2010; Published: December 22, 2010

 

[jeffrez]

Hi Mr. Kite, there might be a number of reasons. They didn't use XMRV positive human controls to validate their methods. Their antibody methods were from a prior zero zero study, and validated using controls in yet another zero zero study. The goat serum controls were probably from recently infected goats. Why does everyone forget/ignore that the virus disappears from the blood after a number of days? Their antibody tests were to directly find XMRV using XMRV targeted manufactured antibodies, they did not look for natural antibodies to XMRV.

Thanks for the explanations. I'm having a hard time believing antibodies wouldn't be detected if they existed in the samples, but I suppose it's possible their methods were flawed. There always seems to be an "out" to keep the yea-sayers going (and the money flowing in to WPI, et al. ;-)), but hopefully it will be sorted THIS year. Although perhaps that's as overly optimistic as expecting it to have been sorted this past year. Once that gravy train gets rolling, I'm sure it must become very compelling for these researchers to maintain their "rivalry" for as long as possible.

 

[omerbasket]

The original Science article study stated the T and B cells were cultured for 42 days. She can't change that to 45 days now, other labs should be able to find what she found the same way she found it. And even then, they now must run the more robust contamination studies, which WPI should also be doing as well, given the results of the Retrovirology studies. If WPI does not re-run their samples with the more robust contamination studies, they will lose credibility in the scientific community.

Kurt, I know you are against XMRV and the WPI - but when Judy talked about 45 days of culture in norway, she did so when she presented it to patients, and she probably just rounded the number up. Anyway, what they really are saying is that you need at least 21 days of culture, and at most 42 days.
WPI did almost everything that can be done in order to check for contamination. All of those papers about contamination talked about PCR. But the WPI didn't use only PCR in their study. They used a number of methods, and they showed an immune response to the virus - and all of those papers have chosen to ignonre it.

 

[omerbasket]

I'm bringing here some of Gerwyn's stuff from the other forum - and you can all see for yourselves why this study, again, says nothing at all:

Hohn et al used primers which have never been able to detect wild type xmrv. This study showed that their PCR could tetect xmrv clones many orders of magnitude higher than would be the case in human hosts. Likewise their serology assay can only detect clone proteins at massive concentrations.Yet again we have researchers ignoring the scientific method and not establishing the diagnostic sensitivity of their assays despite clearly having the means to do so. Any complete clone can infect human cells in vitro whether they are replication competent in vivo or not.Indeed endogenous retrovirus can be made to look infectious using this method!!

They have merely repeated the methods which other research has shown to be inneffective and failed to detect xmrv last time they were used.They have done so despite published evidence that PCR (even with the correct primers) is by far the least sensitive method of detecting XMRV.It is difficult to understand such an obvious departure from the scientific method by professional scientists

About not using the technique that lead Hohn to find XMRV in respiratory tract secretions:

why abandon a technique proven to be able to detect xmrv in favour of a technique which has not. in fact the technique used has now failed them three times

•Oliver Hohn and colleagues wanted to show that they had not discovered xmrv, in god knows whatever patients they actually diagnosed with cfs, even though they had amplified their samples[/color[/size]
•]I wondered why they had activatedPMBCs via pha and then cultured in IL-2
••Well if you do that the pmbcs produce massive ammounts of LAK T cells which in 7 days would have killed xmrv stone dead and denatured any viral dna so in effect it was worse than the methods of McClure
•they determined the sensitivity and specificity of their new elisa assay against friend mulv and mulv gag!! but implied they were specific xmrv proteins so they might have been specific and sensitive for the former but not for the virus they were supposedly looking for!!
•they referr to their earlier ELISA assay as validated when it was calibrated against synthetic dna and vp62 in a study involving Switzer andOliver Hohn which of course could not find wild type xmrv at all
••Oliver Hohn claimed that they had no wt virus available for protein manufacture and calibrate their pcr yet he was in a study that found it in people 6 months ago[/color[/size]
•]Oliver Hohn claimed that they carried out their nested pcr in the same way as urisman and switzer .the latter study also involved him. he does not say that he chose to use the primers of switzer which did not find the virus instead of those used by urisman that did[/color[/size]
•]the serology assays that have discovered xmrv proteins are IHC FISH and WESTERN BLOT[/color[/size]
•]OLIVER HOHN however continues to persist with NOVEL ELISA assays which have never demonstrated any capability of detecting XMRV proteins in a clinically positive sample in favour of other methods that repeatedly have
If he wanted to amplyfy the sample why did he not use the reverse transcriptase PCR which he knew worked and would not have run the risk of destroying viral DNA and laying him openm to the charge of being either duplicitous or a total idiot!

I keep mentioning Oliver kohn because he appears in all the studies mentioned and appears to be something of a common denominator

Remember the cdc sent their samples to him to collaborate their negative serology using a method that had not detected XMRV proteins either!

 

[eric_s]

I don't care about that kind of negative studies too much anymore. I'm waiting for BWG results or work by those people who have reported to be able to find XMRV like Alter, Lo, Hansen and hopefully Singh (the WPI too, of course, but we already know that). As long as we don't get a negative study in a journal like Science, Nature etc. it does not change much for me.