From Professor Vincent Racaniello's ‘Virology Blog', 4 May 2011.
Since the first association of the retrovirus XMRV with chronic fatigue syndrome in 2009 in the US, subsequent studies have failed to detect evidence of infection in patients from the US, Europe, and China. These studies were potentially compromised by a number of factors, such as differences in patient characterization, geographic locations, clinical samples used, and methods used to detect the virus. These and other potentially confounding conditions have been addressed in the most comprehensive study to date on the association of XMRV with CFS.
In the introduction to their paper, published in the Journal of Virology, the authors note other problems with many of the studies of XMRV in CFS patients:
* Too small control populations
* Patient and control samples collected at different times
* Investigators generally not blinded to sample identity
* PCR assays that rely on conservation of viral sequence mainly used
* Limits of detection, reproducibility, and precision of assays unknown
* Controls for each step that would identify analysis not done
* Insufficient numbers of negative controls included
* No study included positive samples from the original 2009 patient cohort of Lombardi et al.
To address these issues, the authors collected blood from 105 CFS patients and 200 healthy volunteers in the Salt Lake City area. One hundred of the patients fulfilled both the CDC-Fukuda and the Canadian consensus criteria for diagnosis of ME/CFS. The patients were selected from a clinic that specializes in the diagnosis and management of CFS and fibromyalgia.
New blood samples were also collected (by a third party) from 25 patients from the original study by Lombardi et al. The samples were blinded for subsequent study. Detection of viral nucleic acids was done using five different PCR assays. Anti-XMRV antibodies in patient sera were detected by ELISA. Finally, virus growth from clinical specimens was attempted in cell culture. The authors used the multiple experimental approaches reported by Lombardi and colleagues.
Let’s go through the results of each assay separately.
PCR for viral nucleic acids. Four different quantitative PCR assays were developed that detect different regions of the viral genome. The assay for pol sequences has been used by several groups and is the most specific PCR assay for XMRV. Three other PCR assays were also used that target the LTR, gag and env regions of XMRV DNA. The pol, gag, and env assays could detect at least 5 viral copies of XMRV DNA. The precision and reproducibility of the PCR assays, as well as their specificity for XMRV, were also demonstrated. DNA prepared from white blood cells of 100 CFS patients and 200 controls were negative for XMRV. For every 96 PCR reactions, 12 water controls were included; these were always negative for XMRV DNA.
XMRV antibodies in human sera. To detect XMRV antibodies in human serum, a portion of the viral envelope protein, called SU, was expressed in cells and purified from the cell culture medium. The SU protein was attached to plastic supports, and human serum was added. Any anti-XMRV antibodies in human sera will attach to the SU protein and can subsequently be detected by a colorimetric assay (we have discussed this type of assay previously). This assay revealed no differences in the amount of bound human antibodies for sera from CFS patients or healthy controls. Some of the patient sera were also used in western blot analysis. Recombinant XMRV SU protein was fractionated by gel electrophoresis. The protein on the gel is then transferred to a membrane which is mixed with human serum. If there are anti-XMRV antibodies in the human serum, they will react with the SU protein on the membrane, and can be detected by a colorimetric assay. When rabbit anti-XMRV serum was used in this assay, the SU protein was readily detected. None of the human sera analyzed by this method were found to contain antibodies that detect SU protein.
Infectious XMRV in human plasma. It has been suggested that the most sensitive method for detecting XMRV in patients is to inoculate cultured cells with clinical material and look for evidence of XMRV replication. The XMRV-susceptible cell line LNCaP was therefore infected with 0.1 ml of plasma from 31 patients and 34 healthy volunteers; negative and positive controls were also included. Viral replication was measured by western blot analysis and quantitative PCR. No viral protein or DNA was detected in any culture after incubation for up to 6 weeks.
Analysis of previously XMRV-positive samples. Blood was drawn from twenty-five patients who had tested positive for XMRV as reported by Lombardi et al. These samples were all found to be negative for XMRV DNA and antibodies by the PCR and ELISA assays described above. In addition, no infectious XMRV could be cultured from these 25 samples.
Given the care with which these numerous assays were developed and conducted, it is possible to conclude with great certainty that the patient samples examined in this study do not contain XMRV DNA or antibodies to the virus. It’s not clear why the 25 patients resampled from the original Lombardi et al. study were negative for XMRV in this new study. The authors suggest one possibility: presence of “trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies”. I believe that it is important to determine the source of XMRV in samples that have been previously tested positive for viral nucleic acid or antibodies. Without this information, questions about the involvement of XMRV in CFS will continue to linger in the minds of many non-scientists.
At the end of the manuscript the authors state their conclusion from this study:
Given the lack of evidence for XMRV or XMRV-like viruses in our cohort of CFS patients, as well as the lack of these viruses in a set of patients previously tested positive, we feel that that XMRV is not associated with CFS. We are forced to conclude that prescribing antiretroviral agents to CFS patients is insufficiently justified and potentially dangerous.
They also note that there is “still a wealth of prior data to encourage further research into the involvement of other infectious agents in CFS, and these efforts must continue.”
Clifford H. Shin, Lucinda Bateman, Robert Schlaberg, Ashley M. Bunker, Christopher J. Leonard, Ronald W. Hughen, Alan R. Light, Kathleen C. Light, & Ila R. Singh1* (2011). Absence of XMRV and other MLV-related viruses in patients with Chronic Fatigue Syndrome. Journal of Virology : 10.1128/JVI.00693-11
Abstract can be found here: http://jvi.asm.org/cgi/content/abstract/JVI.00693-11v1
Same old. Unvalidated novel assays, no replication, and use of a clone. There was also only 2 people retested who were found positive in Lombardi et al. One was the source of a full length clone and EM of the budding virus.
http://scienceblogs.com/erv/2011/05/xmrv_and_chronic_fatigue_syndr_21.php
ERV is a student and is repeatedly wrong. The novel assays in this study have never detected a positive.
Granted. Was interested in finding other opinions ahead of more from those actually involved.
I see such information is now coming out in the articles I have linked to below, and in other US sources.
What I was after was more detail from Singh, Mikovits and others – it seems that this is now coming out.
Be interested, what you make of it JT – you seemingly being more of a ‘scientist’ than moi.
Personally, I await Lipkin to pull a rabbit out of the hat. The story isn’t over. There remains – as Singh states – the virus that infects the prostate cancer and it will be interesting to hear her take on that.
Although I see Switzer had research published last week that couldn’t find XMRV there either?
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019065#abstract0
Still, let’s hear from Singh some more about this one, hey?
Sorry. I see Lipkin has written to Dr Racaniello:
http://www.virology.ws/2011/05/06/ian-lipkin-on-xmrv/?utm_source=feedburner&utm_medium=email&utm_campaign=Feed%3A+VirologyBlog+%28virology+blog%29
My links to the Trine’s article is also posted there too, by ‘GOB’ in the comments. Boy you gotta ‘love’ that guy, GOB I mean. He/She has so much ‘verve’ 🙂
Racaniello:
‘Dr. Lipkin is not saying that the Singh study is ‘worthless’ – he is merely stating that no number of studies will convince non-scientists that the Mikovits study was incorrect. He is saying that it is up to Mikovits and Alter to repeat their studies in an appropriately powered manner to resolve the issue. I’m not sure that will happen. As I’ve said before, the Singh study is extremely well done in all ways. For scientists, there is no confusion about the findings.’
From the above link.
An interview with Singh from CFS Central:
http://www.cfscentral.com/
CFS Central: In your new study it says that “2 positive controls were also included” (line 349 of your study). Were the two positive controls clones or clinical samples from prostate-cancer studies or from Mikovits’s positive CFS patients?
Singh: The samples from Mikovits’ patients were all tested in a completely blinded fashion. We did not know which of them were positive, so could not use them as positive controls. But more accurately, there are no real patient ‘positive controls’ for XMRV. In order to use patient samples as controls, you’d have to first be absolutely certain that these patients have XMRV. How could you do that right now? So we used what you call a ‘clone’ for our PCR studies. But remember, this clone was isolated from a patient (a prostate cancer patient). And this is over 99% identical to the isolates from CFS patients described in Lombardi et al. For the viral culture studies, we used very small amounts of titrated virus that was grown in the lab as positive controls. And all of these positive controls were always positive.
CFS Central: You grew XMRV in the prostate-cancer cell line LNCaP and the breast-cancer cell line MCF-7 in your study “Raltegravir is a potent inhibitor of XMRV, a virus implicated in prostate cancer and chronic fatigue syndrome.” It’s unclear to me why the new study didn’t culture XMRV from those two positives.
Singh: Yes, we did. And these grew just fine. Apologies for not being clearer in the paper. None of the patient samples tested positive, but the positive controls were always positive.
CFS Central: Some patients on the forums find this sentence from the new study problematic: “We are forced to conclude that prescribing antiretroviral agents to CFS patients is insufficiently justified and potentially dangerous.” The Science and the Alter/Lo studies have reported XMRV and related MLVs in CFS patients. CFS patients are very ill, many for decades, and there are no approved treatments. They believe the decision to try antiretroviral drugs should be between a patient and his or her doctor. Why did you feel the need to put that statement in the study and press release?
Singh: The patient and their doctor did not make the decision to try antiretroviral drugs in a vacuum. It was based on reports of finding XMRV in CFS patients. We are now convinced that there is no XMRV in CFS patients–so the reason for starting those drugs does not exist. And there is no good evidence for continuing to use drugs that could lead to serious side effects of liver or bone-marrow failure.
‘…No XMRV protein or DNA was detected in any of the cultures. [emphasis added]’
My question is this: Is the bolded sentence correct, or did the 2 positive controls grow in these cultures and/or did the positive samples continue to test positive in culture? If the positive controls didn’t grow or if the positives controls didn’t test positive in culture, why didn’t they? You grew XMRV in the prostate-cancer cell line LNCaP and the breast-cancer cell line MCF-7 in your study “Raltegravir is a potent inhibitor of XMRV, a virus implicated in prostate cancer and chronic fatigue syndrome.”
Singh: That was our poor wording in the paper (the words in bold). All positive controls grew XMRV–as one would expect. None of the samples from the healthy controls or CFS patients grew any virus in culture. And of course the negative controls did not grow any XMRV.
Using a very astute line of questioning, Mindy Kitei effectively exposed the inadequacies of Dr. Singh’s study and the erroneous / overstated conclusions in the Shin et al paper.
Dr. Michael Snyderman commented on Dr. Jamie Deckoff Jones’ blog:
Dear Friends,
I am proud to see so many intelligent, sophisticated and well informed comments from our group. Some CFS patients have been shaken by the Shin et al paper. Not me. I googled the background of all the investigators and they are all qualified in their own areas. They are lab technicians, experts in nerve transmission and of course, a pathologist. The laboratory listed is ARUP Laboratories which as per the official web site does the same assays available through Quest and LabCorp.
Compare Shin et al’s backgrounds to Drs. Lombardi, Ruscetti and Mikovits who are elite scientists with specific training and a track record in this area of research. Actually, there is no comparison!
Dr. Singh is a respected research pathologist and I look forward to her continued investigation of various cancers for XMRV. Heaven knows why she switched course and decided to write a paper about CFS and XMRV. The methodologies required are way too sophisticated for anyone to do well without a large knowledge base and experience which her group could not possibly have. What should have been concluded is that they did not find or more honestly, that they could not find evidence of XMRV in the context of CFS. This a lot different than saying that it wasn’t there which is not a scientifically correct conclusion for her study.
As someone who has benefited from taking ARVs, I find it ludicrous to be told that I never should have. Agreed that ARVs, at least not the ones that we have may not help everyone, but that is not the point at all.
Keep the faith.
Michael Snyderman, MD