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
Saying you are not crazy has never done anyone any good. Stick to the scientific facts.
Everyone else knows.
@e0eddb636ed2ccc32d389f3684bf35f4:disqus This is a virology study. It has nothing to do with PACE. Period.
I know of a few negative studies where the “psychiatric brigade” was involved, but most of them were not. Virologist have no incentive to cover up a retrovirus. It would be like Howard Carter saying
“I have seen enough tombs for today, lets leave this one of King Tut alone, it can’t be that important.”
This is through multiple methods. The CDC retested 20 samples, they found nothing.
The rest of your comment is not scientific. This is why they lowered the stringency of the primers.
The WPI has not declared those samples positive in a published study. If you being to accept data from unpublished work then you also have to accept the finding of the other viruses by the NCI, WPI, Hanson, Lo, Alter. This is evidence that supports the association.
Also important is to lower annealing temperatures, to widen the virus variables (as is usual in wild viruses) detectable. This study under discussion did not replicate Lombardi, yet again. This is getting rather boring.
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This is the reason we want the WPI to gain Chase Community Giving Funding (don’t forget to vote on facebook, 19-26 June) and for the patient funding initiative Count ME In! to grow. The WPI know how serious the disease is, they have the lab and clinic ready, and with the funding could begin trials, whether of Anti Retro Virals or (for the many co-infections) Anti Virals, and other hopeful therapies, while tracking progress via viral load tests and cytokine tests. This is the research that is desperately needed, and has been missing for all these years.
This is where ME research dollars, pounds and euros should go.
To avoid confusing ME with CFS, the disease we’re discussing is Myalgic Encephalomyelitis (ME for short). CFS was invented by the CDC to make ME sound less serious. ME has probably been around hundreds of years but not discernable above the background of TB, syphilis, black death, and many other serious diseases.
The WPI have, using several different techniques, found XMRV in 98 or 99% of their ME patients – how can the pyschs & other deniers deny a “link� And the WPI have never claimed a causal relationship – but that firm link indicates this, to anyone of modest intelligence.Those who have read the obligatory tome “Osler’s Web†will know the facts. Dr Paul Cheney, around 1985, took some of his ME patients’ MR scans to an MRI expert, who showed him a set of identical scans – from AIDS patients. (see the videos on YouTube). Maybe they hadn’t sorted out the retroviral origin of AIDS at that time, but the penny will have dropped later. So we then knew, back then, that a retrovirus was involved.
Then in 1991 the Wistar Institute researcher Elaine DeFreitas actually found a retrovirus in her ME patients, even showed electron-micrographs of the virus inside mitochondria where they disrupt the mitochondrial function of producing energy in the cell – hence the weakness or fatigue felt by patients. She couldn’t continue with the research as her funding was promptly withdrawn.
With all the government institutions turning their back on the biomedical cause of M.E., it was left to the “private sector†to fill the deficit. The Whittemore family, with their daughter Andrea an M.E. patient, engaged retrovirology experts including Judy Mikovits and established a private research centre, the Whittemore-Peterson Institute (WPI). Judy already suspected a retrovirus was involved and set out to prove it to the high standards of the journal “Science” (not to confuse with “Science magazine”).
The rest is history. Several ME patients have started on ART’s (anti-retroviral therapies) and are blogging about their experiences – most are improving. The drugs are based on those developed for treating AIDS patients – successfully. However XMRV is not the same as HIV and it may be possible to develop special drugs for XMRV which will be more effective – if the drugs companies remove their collective skulls from their colons and get on with the job. With perhaps seventeen million people worldwide affected, this could be very profitable.
— Laurence Swift (retired vet)
The WPI are invested in finding the cause and cure for neuro-immune disease. If they turn out to be wrong about Human Gamma RetroViruses, then they will use their considerable expertise, understanding and facilities to look elsewhere, I am sure. But the HGRV theory of causation has not been disproved at this point in time.
This is why there is nothing wrong with the patient statement quoted by Roy Hobbs.
Hi Tom. One of the WPI patients used in the Singh study was the source of the EM image of viral budding. This person’s sample was found negative by Singh et al.
@50abda0d1c529d41345152ecc4d7cf6f:disqus To avoid confusion stick with CFS, or ME/CFS. All recent biomedical research mentions CFS, not ME. There are no published studies on XMRV and ME, not on spinal fluid an ME. The Canadian Consesus Criteria mentions ME/CFS, not ME. Was it Byron Hyde who said that the ME in ME/CFS is not the ME of the ICD and that probably only a subset of the ME/CFS patients have ME?
Bad time for a name change now that finally some serious biomedical research is taken place for this disease called CFS. Scientist who have got involved or interested in this disease over the past year and a half refer to it as CFS.
I refused that test, because it is even more questionable than the commercial tests from VIP Dx. No publications whatsoever on the nature of test. I refused for several reasons, one of them because it is a PCR test and that was just after the publication of the contamination papers.
But i think we should use “ME/CFS” and not “CFS” anymore. This is what the CFSAC has recommended, what is used in the Canadian Consensus Criteria and what the NIH are using.
It is a human gamma retrovirus, thus a HGRV.
@Gob987:disqus Did you ask them directly instead of relying on the rumour mill?
Are you a scientist yourself? Because I’m starting to read the same types of vague objections being parroted over and over again in rote fashion. You don’t seem to understand that many of the same potential reasons you cite for Singh et al to find nothing would also lower the chances of Lombardi et al to consistently find those positive results.
Now you are being ridiculous and you know it. As I said before, you sound like a desperate defense attorney doing anything possible to create a reasonable doubt. If your goal is to defend the WPI results and XMRV at any and all costs, that would be understandable. Otherwise, it does not.
could it be that the viral budding was from another virus?
What do you think about CFS Dr. Racaniello? Do you think there’s sufficient evidence to warrant further research regarding infectious agents & CFS? Given that “Science” is apparently reputable – what mistakes did they make in publishing the original paper which has only accomplished further studies to disprove it and stir up unending controversy? Your opinions would be greatly appreciated and respected. Thank
so your saying that the virus photo of xmrv, isnt even xmrv ?? its another virus, someone better start deleting all those photos on the internet of xmrv then.
They are the same assays as used by VIP Dx. The stomach biopsy is a for a research study.
so if everyone is saying this is not a real virus in cfs, are the wpi breaking the law selling tests for a virus that isnt in humans ?
the photo of the virus is from a cfs patient, if that virus is not from that patient is that breaking the law?
Cant wpi film dr mikovits taking a virus out of a patient sample?
You know what this could go on forever! there could be a few thousand people for the next 100 years who are left thinking they are xmrv positive as they were told so.
what a mess.
its 2011 right?
You are mistaking scientific data for objections. The reason for the repetition is understandable when many are confused and repeatedly ask the same questions, whilst not being in possession of the data. Please state what you suggesting would lower the ability to detect the virus in Lombardi et al and I will explain.
Patients who have tested positive for strains at the WPI other than XMRV would not have been detected in this study. Either we are accepting only published data or we take into account data presented at workshops or even data collected through private testing. Regardless, unvalidated assays cannot be used to claim a person is negative.
Hi Justin,
I empathize with your frustration. To avoid long hair-splitting “explanations” of the differences between CFIDS ME CFS FM and so on… By now we SHOULD get that anyone posting here is referring to the same thing – or at least feels sick in the same way. The points I wanted to make is why we are all prone to referencing a comment a University of Miami researcher once made comparing Aids patients to ME CFS… patients. It seems she was trying to provide some sort of context – as the general public and %90 of the medical profession apparently can’t comprehend the level of severe suffering and utter sickness (there must be some widespread deafness too, because we certainly do tell them) involved with having ME CFIDS CFS FM…. That said, unless the HIV + person is in an underdeveloped nation (as we all are regarding CFS) once a person has actually developed AIDS it’s very unlikely they’d be untreated. Sadly by the time their status is AIDS they’ve developed one of the associated cancers or identifiable infections. I tend to think one of the reasons the establishment feels content about allowing the suffering of CFS ME patients to continue is that it does not (many of us would say “unfortunately”) result is a rather quick and visibly dramatic death. They must also feel they have certain data concerning how this illness is spread – and as we can all easily understand it’s very easy to keep the public content with references to psychosomatic disorders and the like. They could not very well do that with the hundreds of thousands of men suddenly wasting from weight loss, with caner lesions all over them, dying by the thousands in multiple hospitals from San Francisco to NY. I suppose we are collectively making some progress though – at least today scientists are fighting with one another over this. They tend to have enormous ego, so while they do research to disprove one another and improve one over the other, we will eventually get an answer. I don’t think HIV patients really care that their suffering was sorted out via fighting egomaniacal scientist in France and the USA for their own ends, neither should we. The fact that the Whittemore P XMRV study MAY have been wrong and “Science” made a huge blunder in publishing it is the best thing that could have happened! I hope it becomes more controversial between them each day and more and more universities and labs jump on the bandwagon – who cares if it’s for the wrong reason if it means treatment and a life for us!
This is in the published paper.
They sequenced a full length clone of XMRV from this patient.
OK, Eric, we can follow the crowd and use the composite term ME/CFS. Or better, ME (CFS). With the ultimate aim of reverting back to the original term ME, historically and scientifically correct. The term “CFS” in unheard-of in the general population of the UK who all say “ME”, and I believe this applies in Australia & other ex-colonies. Only the medical profession have ever used the term “CFS”. Even the WPI uses “ME”, variably.
All the old papers calling it “CFS” – like our PACE Trial – have used the term to cover a range of patients with differently-defined symptomatologies. It’s really time we relegated all these confusing studies to history and went ahead with the right name and the right definition.
perhaps dr. racaniello could comment on the full-length clone?
This is a human retrovirus. Both the CDC and committee for emerging infectious diseases in the UK accepted it was several months ago.
There is no evidence for contamination and negative studies using unvalidated assays do not suggest differently.
Again, using your own logic, Singh data is obviously published in the Journal of Virology. Therefore it should be acceptable. But since it is negative, albeit published, you say oh but it’s unvalidated?
There also appears to be a discrepancy regarding the 14 samples tested that came from patients referred by WPI. Were they in fact repeatedly positive when tested by WPI? Yes or no? Whether these 14 were published upon or not makes no difference, unless you are playing defense attorney again.
So Nested PCR and Nested RT PCR are two totally fifferent things, but on the other hand it’s perfectly acceptable to use the term Nested PCR when you really did Nested RT-PCR (which, again, is totally different? Rrright….
Second, how would you even know for certain that Singh didn’t also use Nested PCR when she meant Nested RT-PCR? She in fact states that she has used the same assay as in the original studies…
Are you a scientist or not? If so, please define your background and experience in running these types of assays. If not, I’m sorry but I am not interested in your explanation. Unfortunately, often non-scientists tend to be very selective in what they learn, in order to support their pre-existing conclusions.
Please give us your definition of “evidence” (in science).
As a patient who has actually experienced a certifiable level of chronic fatigue [To: Dear Impatientpatient, I am giving you an order not to go signing ‘on the dotted line’ anywhere until I clear you to do so. Also, you know that jury duty summons you showed me? Well, I will personally write the judge to get you out of it. What is the reason for this directive, you ask? Basically, you are too fatigued to think straight might do something stupid, and it will only get worse until your scheduled treatment. From: Dr.
Icareaboutyouandallthegraymatterthatyoucontinuouslyobservespewingfrommyheadactuallyknowswhatitstalkingabout] I’d like to say I hope one way or the other this latest study brings this whole issue some closure. But after reading the comments here that seems pretty doubtful.
Anyway, I’m wondering… Is there anyone willing to do a study on whether Oppositional Defiant Disorder (ODD) is a symptom of Briquet’s Syndrome (otherwise known as Somatization disorder)? How about Hypochondria?
I believe the results of the analysis simply suggest looking at other possibilities for pathology : We examined cerebrospinal fluid from 43 CFS patients using polymerase chain reaction techniques, but did not find XMRV or multiple other common viruses, suggesting that exploration of other causes or pathogenetic mechanisms is warranted.
But what about critical thinking?
Yes, it is acceptable, because after reading the study you would know that the conditions are for Nested RT-PCR, as they say so in the paper. Shin et al makes no mention of Nested RT-PCR, so it is only Nested PCR. Why would you leave out the RT part if you actually undertook RT, you would not.
If you have no scientific data to support your personal beliefs, it is better to admit that.
Yes, I would agree you are being selective.
Well it will be terrible for the thousands who’d prefer to be convinced they have XMRV if and when it’s disproved and actually turns out to be VRXZSHCG for which treatment is available. Perhaps those fantom people are the one’s whose CFS would respond well to Prozac – I should hope so anyway!
Validation of an assay to be able to identify clinical positives is not debatable. Those assays have not been validated.
2 were from patients from Lombardi et al.
12 were from unpublished experiments.
As the assays are not validated, than the results are meaningless. What variety of HGRV did the other 12 test positive for? It is unscientific to gloss over this. The diversity of the virus is enormous.
Firstly, any contamination claim must be able to account for all the observations. None proposed have done this.
And why would you state that you did the dame PCR as the original investigators when you did not?
See how easy that was?
ee
@RRM
Yes, it is acceptable, because after reading the study you would know that the conditions are for Nested RT-PCR, as they say so in the paper. Shin et al makes no mention of Nested RT-PCR, so it is only Nested PCR. Why would you leave out the RT part if you actually undertook RT, you would not.
It’s not debatable but it has never happened in the history of science that a validation study used the results of the study it was trying to validate in order to calibrate their assays to, in order to validate the original finding.
Please prove me wrong.And it was Judy Mikovits who chose those patients, because she felt they had the biggest chance of “success” in Singh’s hands. You are now implicitly accusing Mikovits of being a lousy scientist for providing these samples with ‘too much diversity’.
@a76cf1733deb438a3e6b339a2f230d8a:disqus
The posts above on the right.
Several papers have claimed to have used the same methodology, they did not. Perhaps for the same reason you have not known what the PCR type was, you either did not read the paper or did not try to read it throughly.
And what if…….there would actually zero positives to detect?
Now, I know this isn’t the case with XMRV given the mounting evidence you guys find on the forums, but take another finding like this, but then one that is false (again, unlike XMRV).How exactly would your scientific methodology *solve* this false finding? By going dizzy running in circles?
Again, try getting that passed the regulators. I suggest you start doing some basic research of your own into this matter.
Are you seriously now saying that the diversity of the virus is a problem. Congratulations, you are starting to understand.