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 14 patients from the original study by Lombardi et al. The samples were blinded for subsequent study. Detection of viral nucleic acids was done using four 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. These 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.
Presence of mouse DNA. After not finding XMRV using qPCR, serological, and viral culture assays, the authors used the nested PCR assay described by Lo et al. Although positives were observed, they were not consistent between different assays. This led the authors to look for contamination in their PCR reagents. After examination of each component, they found that two different versions of Taq polymerase, the enzyme used in PCR assays, contained trace amounts of mouse DNA.
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 14 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
I would like to thank Vincent Racaniello, Alan Dove, and others for their time to explain and discuss the studies on XMRV. I really appreciate it. Keep up the good work on this blog and TWiV.
I hope you will invite Ila Singh back on TWiV to discuss her latest research.
I am wondering. What does this study mean for the XMRV – prostate cancer studies?
Do I remember correctly that Ila Singh wasn’t only looking for XMRV in blood but also in organs to find the reservoir?
Test accuracy is determined by sensitivity and specificity.
Sensitivity is how many cases of a disease a particular test can find.
Specificity is how accurately it diagnoses a particular disease.
Those looking for only what matches VP62 will miss what is not VP62.
Good you agree that it is illogical for the CDC to have switched assays. It is.
There is no evidence for contamination in Lombardi or Lo et al.
Your interpretation of why they changed assays is one thing, the fact that they did cannot be disputed. It is on the blood working group slides.
It would never get passed regulatory authorities. This is how it works. Then again you don’t know what the methodology in Lombardi et al was.
Lomardi et al used Nested RT-PCR, not nested PCR.
Lombardi et al used Nested RT-PCR, not Nested PCR. Perhaps read the paper next time.
This is how it works, there is no other way to validate an assay.
Thank you for demonstating that you don’t even know what the work replication means.
Lombardi et al validated their results with 4 methods and retesting at the Cleveland clinic and NCI.
They used a clone.
You cannot disprove a studies results if you do not use a validated assay. Negatives prove nothing.
Whereas Frank Ruscetti is one of only a handful of people who have discovered a human retrovirus. Appeals to authority are not science. Please stick to the facts.
The NCI tested 20 samples.
Replication is the easiest way to disprove.
I think you very clearly showed the problem in this debate with your opening sentence. Ila Singh is on your side when her results match WPI. She’s on the other side when they don’t. The only “side” that should matter in this whole debate is the side of truth, the side of accuracy. That is what science is all about. The fact that people like you see Dark Forces and conspiracies everywhere, and will instantly denigrate any scientist and any study that does not support your position speaks volumes. Oh, and having your kid stay on oral tetracycline for over a year for acne is a very bad idea, and I can tell you this from personal experience.
I can’t actually reply to your latest comment above, Gob, but RT-PCR is a variant of PCR. Saying that one didn’t do PCR when one did do RT-PCR is thus incorrect (of course, the same applies to nested PCR).
Remember, if nested PCR does not also include nested RT-PCR, the authors of Lombardi et al. would be not telling the truth multiple times (see the quotes from the Lombardi paper above).
When he says “on our side” I’m pretty sure he means on the side of bona fide science (ie accuracy, the truth); that’s the side that ME patients are rooting for, in my experience. His further remarks in the post point in that direction too.
and yes, there are dark forces and conspiracies in the world of ME ‘science.’ That is a fact.
Thank you. And when the original WPI results are clearly and completely invalidated in the next few months, as I now expect they will be, can I expect another sincere and complete apology and admission that you were wrong as well? I will certainly offer the same to you if WPI is indeed vindicated.
As someone else said, the fact that this team would have preferred to find XMRV makes this paper even more persuasive.
And these are very good scientists who did this study. However, they are human. They made at least one error in drafting the paper. It is possible they made other errors. My point is, the fact that they didn’t find XMRV doesn’t mean we know with certainty that ME is unassociated with XMRV.
from WSJ blog:
http://blogs.wsj.com/health/2011/05/04/study-finds-no-link-between-xmrv-and-chronic-fatigue-syndrome/
“The Whittemore Peterson Institute’s Judy Mikovits, who led the research team on the Science paper, tells the Health Blog she has not yet read the complete study. She says XMRV isn’t fully understood. And she also says that one of the statements in today’s paper is incorrect. She tells Health Blog that not all of the 14 people who previously tested positive for XMRV were part of the original Science paper; only two of them were.”
Au contraire, you (and your fellow forum scientist genius Gerwyn) didn’t know what the methodology in Lombardi et al. was, as you were clinging on to the thought that the 67% results was obtained using other assays than “just” nested (RT-)PCR for gag.
See for instance this misguided post by you about the Lombardi methodology:
http://www.mecfsforums.com/index.php/topic,4984.msg55023.html#msg55023
You: “The figure of 68% [sic] was reached through multiple methods, not all were tested with every method”.
Lombardi: “we isolated nucleic acids from PBMCs and assayed the samples for XMRV gag sequences by nested PCR. Of the 101 CFS samples analyzed, 68 (67%) contained XMRV gag sequence.”
..and Gerwyn’s post:
http://www.mecfsforums.com/index.php/topic,4984.msg55115.html#msg55115
Question: “a) Please give me the quote from the study (or the addendum) were it says that the 67% figure was reached after culturing the samples for 42 days”
Gerwyn: “it is in the study omer just read through it.”
And please give up the meme about regulatory authorities. No one has ever confirmed a finding using your proposed methodology, that subsequently went through regulatory authorities. And you know it.
No they are different methodologies. Nested RT-PCR is not the same as Nested PCR. To not even understand this is quiet amazing.
And how would he know “bona fide science”? Why, it’s the science that supports “our side”. I have no doubt that this same Ila Singh and her studies were considered true and accurate up until a few days ago by your side. And the almost casual approach to trying powerful ARV meds as if they were simple antibiotics is kind of frightening.
This latest snippet from you is really problematic instead of helpful to your argument:
If XMRV is really THAT hard to find (assuming the Lombardi results are correct), not only through PCR assays but also through serology and culturing, there is no way that all those four methods worked AND that two independent laboratories confirmed the WPI’s results.
When you think about it, the multiple confirmations from multiple laboratories within a single study really do indicate that something was wrong with those samples used in the study.
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It will sadly not convince you, but here is a quote from an actual PdD:
“If the objective of the study is to find out if Lombardi et al had a correct result (ie whether they had true positives or false positives), then how can their positive samples be used as the basis for the design of a new test? Doing that would assume that those positives had already been validated as true positives, which would defeat the entire point of the study!”
Of course, this proves that this guy is part of the conspiracy, as otherwise he wouldn’t be telling this nonsense that goes in the face of thousands of years of science?
PhD, sorry….
Are you incapable of saying “thank you for the clarification, I stand corrected”?
Do you still feel that the statment:
“Sensitivity is the problem [and not specificity]. VP62 is not representative of HGRV.”
..is correct?
Dear Prof. Racianello
There’s something i’d like to know. Maybe it has already been answered in all those comments, i don’t know.
Dr. Singh has tested 305 individuals in that study. 0 were found positive for XMRV.
In Schlaberg et al. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2739868/#__sec1) Dr. Singh found 4% of the healthy controls to be XMRV+.
I think the chance to get 0 positives is very small if XMRV is indeed in 4% of your 305 samples and i don’t see why people with ME/CFS should carry less XMRV than healthy controls.
Maybe someone can do the math, i would have to back and read up how to do that calculation, but i think the chance for that outcome is so small that we can forget about that possibility.
So, unless there’s a geographical factor and XMRV does not occur everywhere and those studies were not designed in such a way that their results woud not be distorted by this, it’s not possible both are correct. Am i right?
So what do you think about that?
I think Dr. Singh will have to adress that. Will she distance herself from the findings in Schlaberg et al. now?
I would like her to use the assays described in her latest paper on prostate cancer patients she has found XMRV+. Would that make sense?
Thank you
Eric Schmalz
That’s Racaniello, sorry for the typo.
Nature. 1984 Dec 20-1985 Jan 2;312(5996):757-60.
Molecular cloning of lymphadenopathy-associated virus.
Alizon M, Sonigo P, Barré-Sinoussi F, Chermann JC, Tiollais P, Montagnier L, Wain-Hobson S.
Abstract
Lymphadenopathy-associated virus (LAV) is a human retrovirus first isolated from a homosexual patient with lymphadenopathy syndrome, frequently a prodrome or a benign form of acquired immune deficiency syndrome (AIDS). Other LAV isolates have subsequently been recovered from patients with AIDS or pre-AIDS and all available data are consistent with the virus being the causative agent of AIDS. The virus is propagated on activated T lymphocytes and has a tropism for the T-cell subset OKT4 (ref. 6), in which it induces a cytopathic effect. The major core protein of LAV is antigenically unrelated to other known retroviral antigens. LAV-like viruses have more recently been independently isolated from patients with AIDS and pre-AIDS. These viruses, called human T-cell leukaemia/lymphoma virus type III (HTLV-III) and AIDS-associated retrovirus (ARV), seem to have many characteristics in common with LAV and probably represent independent isolates of the LAV prototype. We have sought to characterize LAV by the molecular cloning of its genome. A cloned LAV complementary DNA was used to screen a library of recombinant phages constructed from the genomic DNA of LAV-infected T lymphocytes. Two families of clones were characterized which differ in a restriction site. The viral genome is longer than any other human retroviral genome (9.1-9.2 kilobases).
Then Lombardi et al., according to you, LIED when they stated (several times) that they did nested PCR without mentioning that they actually did nested RT-PCR, which is totally different?
For instance, this statement from the Lombardi et al. paper: “[we] assayed the samples for XMRV gag sequences by nested PCR” is a totally inaccurate statement by the authors?
And, from the supporting online materials:
“To avoid potential problems with laboratory DNA contamination, nested PCR was performed with separate reagents in a separate laboratory room designated to be free of high copy amplicon or plasmid DNA”
I know you have a strange way of arguing, but I would never have thought you would accuse Lombardi et al. of lying or at least incorrectly state their techniques. After all, nested PCR is a totally different methodology from nested RT-PCR and Lomardi et al. stated several times that they did nested PCR.
Oh boy…
In the law we say that a ‘statement against [one’s own] interest’ is more likely to be reliable that a self-serving statement.
Additionally, I am not aware of any history of CDC’s routinely lying about PC as it does about ME; are you?
So you concede that your original assertion, that Van Kuppenveld couldn’t detect XMRV in samples provided to him by WPI, is factually incorrect?
Drum roll please….
The article’s title is a misleading misnomer. It should really be called “Absence of the Ability of Our New Test Protocol to Detect XMRV Even In WPI’s Positive Samples”. That says it all.
The article’s title is a misleading misnomer. It should really be called “Absence of the Ability of Our New Test Protocol to Detect XMRV Even In WPI’s Positive Samples”. That says it all.
This is unrelated to the study; but I’d just like to say, fwiw, I have no interest in medicine, much, much, much less in reviewing scientific papers. I do not have the expertise; it taxes me cognitively; uses up my small reserves of energy and bores me. The only reason I am here on this science blog is because I, and the millions of other ME patients, have been brutally abused by our governments and insurance lobbyists under the guise of ‘science.’
People with ME are more physically disabled than people with *untreated* AIDS, yet we use our small reserves of health and energy to learn and comment on the science, because if we don’t, we will never get any help from our governments, medicine or science and we will just continue to rot and die. That is why there are 200+ comments on this post and zero on most of the other posts on this blog.
As any economist can tell you, it is very much in the interest of the government and the CDC to solve the ME/CFS problem, so I guess that makes their negative findings even more reliable.
Your argument hinges on the idea that CDC is routinely lying about ME/CFS but reading a shitty conspriracy book five times doesn’t make is so. Government bodies want to solve this problem, the scientists involved want it, and while I understand the lack of progress is frustrating, there is zero evidence or motive for this vast conspiracy to hide the obvious (because evident to truth seeking patients on a forum) truth.
Ila Singh has found XMRV in previous studies. So why not now? – and why did she even bother to publish the paper? It doesn’t add anything to science, just another negative paper, to join all the others that refused to use the same techniques as the WPI. I suspect if they DID do a true replication study, they would find lots of XMRV.
Personally I do know a little of “bona fide science” – I gained my Master’s degree with a dissertation on a mycoplasmal disease, after experience treating it as well as doing a bit of lab work cultivating similar mycoplasmas.
Ila Singh has found XMRV in previous studies. So why not now? – and why did she even bother to publish the paper? It doesn’t add anything to science, just another negative paper, to join all the others that refused to use the same techniques as the WPI. I suspect if they DID do a true replication study, they would find lots of XMRV.
Personally I do know a little of “bona fide science” – I gained my Master’s degree with a dissertation on a mycoplasmal disease, after experience treating it as well as doing a bit of lab work cultivating similar mycoplasmas.
Stop all science now!!! This is the best way to avoid contamination. My God RRM you get funnier and funnier by the minute.
There is zero evidence of contamination in any positive study. Infact there is an abundance of evidence of zero contamination. A 0/0 study where the investigater cant even find population levels of HGRV is infact further evidence of a poor assay. It tells us nothing about anything else.
It doesnt matter how many times you say the word contamination it doesnt make it fact. RRM by the way what is your interest in this debate, are you a patient and doctor or a scientist etc?
There is an alternative explanation:
– Singh found that 4% in prostate tissue. Thus, it remains possible that (she thinks that) XMRV is present in tissue but not in blood.
I’m sorry about your attitude to ARV’s. People trialling them at present are apparently having few or no troubles with them. Some of these (like Dr Jamie D-J of the WPI) are doctors themselves – the virus does not discriminate according to status or education. There is plenty of experience with these drugs in people with HIV/AIDS.
If you were reduced to the level of a vegetable and found that there was a drug available that could make you once again at least half-functional, wouldn’t you want to try it?
There is also at least one error in the original Science report. From the authors’ response in ScienceMag:
“We regret that a sentence in the original supporting online material (…) implied that immunological abnormalities were part of the CFS diagnosis”
So it’s equally possible that they made other errors.
I wouldn’t be paranoid if they weren’t out to get me! Seriously, Roy, I did not put my son onto oral long-term lymecycline. He is 21, at University studying biomedical science, and he arranges all his own doctoring.
Nope. All 14 were positive. Only 2 of them were from the Science study, but the other ones were selected by Judy Mikovits, and I am sure that she decided to supply Singh with the most reliable positive patients she could find.
Thus, the fact that Mikovits decided to supply Singh with 2 repeatedly positive “Science” patients and 12 repeatedly positive other patients is actually speaking in favor of Singh’s results and against WPI’s results.
RRM do you even know what the PACE study is? For someone so interested in ME I would be amazed if you havent heard of it. Please enlighten us as to how water tight and well designed the PACE study was and what it proved.
According to the PACE designers and sections of the press it was a water tight design. Does that make it true?
You are talking to people who have followed this issue for years and understand what the scientific method actually is. You have an an almost juvenille and illogical understanding of what the scientific method is. Your desperation to avoid it at all costs is very telling and you may very well fool the naive but you dont fool long standing patients.
Also please offer up a credible hypothesis of what could cause ME and what kind of condition you believe ME/CFS is.
Like many other contamination theorists you consistently push the myth that is is only the WPI who can find XMRV/HGRV when it infact has been validated by the NCI, CC and Alter to name but a few.
There is are no sides only data. Shin et al has used unvalidated assays. There is nothing more to be said.
Yes, exactly. I think we have to find that out. Probably the easier route will be to try the blood assay on XMRV+ prostate cancer cases than the other way round…
But in this case the conclusion that there is no associaction between ME/CFS and XMRV would not be correct at this point. One could only conclude XMRV can’t be found in the blood of ME/CFS patients or healthy controls at this moment, i think.
Yes.
But imho we should focus more on getting “real” scientists to work on ME/CFS than trying to understand it ourselves. We must understand it to some degree, but we can never solve it. I’d rather have every patient donate to good research than paying a lot for experimental treatments and becoming an expert of the disease, but without knowing how to defeat it.
Then please read the following and reconsider your position:
http://www.annals.org/content/126/1/91.extract
The first paragragp will do as it shows where your misunderstanding stems from.
Still, that is what the original study reported.
Of course, we can never be sure that an unknown an undetectable strain of a virus is present is tissue that has not yet been tested. But, using this line of reasoning, you cannot even assert that there is no association with flu and HIV because, for all we know, an unknown strain of HIV is floating around in tissues of people with the flu.
That is exactly why the onus to prove something is on the person making the claim.