The long awaited paper that would ‘solve’ the controversies about the presence of Xenotropic Murine Leukemia Virus-related virus (XMRV) in patients with chronic fatigue syndrome (CFS) was finally published in PNAS last week [1]. The study, a joint effort of the NIH and the FDA, was withheld, on request of the authors [2], because it contradicted the results of another study performed by the CDC. Both studies were put on hold.
The CDC study was published in Retrovirology online July 1 [3]. It was the fourth study in succession [4,5,6] and the first US study, that failed to demonstrate XMRV since researchers of the US Whittemore Peterson Institute (WPI) had published their controversial paper regarding the presence of XMRV in CFS [7].
The WPI-study had several flaws, but so had the negative papers: these had tested a less rigorously defined CFS-population, had used old and/or too few samples (discussed in two previous posts here and here).
In a way, negative studies, failing to reproduce a finding, are less convincing then positive studies. Thus everyone was eagerly looking forward to the release of the PNAS-paper, especially because the grapevine whispered this study was to confirm the original WPI findings.
Indeed after publication, both Harvey Alter, the team leader of the NIH/FDA study, and Judy Mikovitz of the WPI emphasized that the PNAS paper essentially confirmed the presence of XMRV in CFS.
But that isn’t true. Not one single XMRV-sequence was found. Instead related MLV-sequences were detected.
Before I go into further details, please have a look at the previous posts if you are not familiar with the technical details , like the PCR-technique. Here (and in a separate spreadsheet) I also describe the experimental differences between the studies.
- (2010/02/15) Finally a viral cause of chronic fatigue syndrome or not? How results can vary and depend on multiple factors.
- (2010/04/27) Three studies now refute the presence of xmrv in chronic fatigue syndrome (cfs).
- Google Spreadsheet Data XMRV-CFS
Now what did Lo et al exactly do? What were their findings? And in what respect do their findings agree or disagree with the WPI-paper?
Like WPI, Lo et al used nested PCR to enable detect XMRV. Nested means that there are two rounds of amplification. Outer primers are used to amplify the DNA between the two primers used (primers are a kind of very specific anchors fitting a small approximately 20 basepair long piece of DNA). Then a second round is performed with primers fitting a short sequence within the amplified sequence or amplicon.
The first amplified gag product is ~730 basepairs long, the second ~410 or ~380 basepairs, depending on the primer sets used: Lo et al used the same set of outer primers as WPI to amplify the gag gene, but the inner gag primers were either those of WPI (410 bp) or a in-house-designed primer set (380 bp).
Using the nested PCR approach Lo et al found gag-gene sequences in peripheral blood mononuclear cells (PBMC) in 86.5% of all tested CFS-patients (32/37) and in 96% (!) of the rigorously evaluated CFS-patients (24/25) compared with only 6.8% of the healthy volunteer blood donors (3/44). Half of the patients with gag detected in their PBMC, also had detectable gag in their serum (thus not in the cells). Vice versa, all but one patient with gag-sequences in the plasma also had gag-positive PBMC. Thus these findings are consistent.
The gels (Figs 1 and 2) showing the PCR-products in PBMC don’t look pretty, because there are many aspecific bands amplified from human PBMC. These aspecific bands are lacking when plasma is tested (which lacks PBMC). To get the idea. The researchers are trying to amplify a 730 bp long sequence, using primers that are 23 -25 basepairs long, that need to find the needle in the haystack (only 1 in 1000 to 10,000 PBMC may be infected, 1 PBMC contains appr 6*10^9 basepairs). Only the order of A, C, G and T varies! Thus there is a lot of competition of sequences that have a near-fit, but are more preponderant than the true gag-sequences fitting the primers).
Therefore, detecting a band with the expected size does not suffice to demonstrate the presence of a particular viral sequence. Lo et al verified whether it were true gag-sequences, by sequencing each band with the appropriate size. All the sequenced amplicons appeared true gag-sequences. What makes there finding particularly strong is that the sequences were not always identical. This was one of the objections against the WPI-findings: they only found the same sequence in all patients (apart from some sequencing errors).
Another convincing finding is that the viral sequences could be demonstrated in samples that were taken 2-15 years apart. The more recent sequences had evolved and gained one or more mutations. Exactly what one would expect from a retrovirus. Such findings also make contamination unlikely. The lack of PCR-amplifiable mouse mitochondrial DNA also makes contamination a less likely event (although personally I would be more afraid of contamination by viral amplicons used as a positive control). The negative controls (samples without DNA) were also negative in all cases. The researchers also took all necessary physical precautions to prevent contamination (i.e. the blood samples were prepared at another lab than did the testing, both labs never sequenced similar sequences before).
(people often think of conspiracy wherever the possibility of contamination is mentioned, but this is a real pitfall when amplifying low frequency targets. It took me two years to exclude contamination in my experiments)
To me the data look quite convincing, although we’re still far from concluding that the virus is integrated in the human genome and infectious. And, of course, mere presence of a viral sequence in CFS patients, does not demonstrate a causal relationship. The authors recognize this and try to tackle this in future experiments.
Although the study seems well done, it doesn’t alleviate the confusion raised.
The reason, as said, is that the NIH/FDA researchers didn’t find a single XMRV sequence in any of the samples!
Instead a variety of related MLV retrovirus sequences were detected.
Sure the two retroviruses belong to a similar “family”. The gag gene sequences share 96.6% homology.
However there are essential differences.
One is that XMRV is a Xenotropic virus, hence the X: which means it can no longer enter mouse cells (MR= murine (mouse) related) but can infect cells of other species, including humans. (to be more precise it has both xenotropic and polytropic characteristics). According to the phylogenetic tree Lo et al constructed, the viral sequences they found are more diverse and best matches the so-called polytropic MLV viruses (able to infect both mouse and non-mouse cells infected). (see the PNAS commentary by Valerie Courgnaud et al for an explanation)
The main question, this paper raises is why they didn’t find XMRV, like WPI did.
Sure, Mikovitz —who is “delighted” by the results—now hurries to say that in the meantime, her group has found more diversity in the virus as well [8]. Or as a critical CFS patient writes on his blog:
In my opinion, the second study is neither a confirmation for, nor a replication of the first. The second study only confirms that WPI is on to something and that there might be an association between a type of retroviruses and ME/CFS.
For 10 months all we’ve heard was “it’s XMRV”. If you didn’t find XMRV you were doing something wrong: wrong selection criteria, wrong methods, or wrong attitude. Now comes this new paper which doesn’t find XMRV either and it’s heralded as the long awaited replication and confirmation study. Well, it isn’t! Nice piece of spin by Annette Whittemore and Judy Mikovits from the WPI as you can see in the videos below (… ). WPI may count their blessings that the NIH/FDA/Harvard team looked at other MLVs and found them or otherwise it could have been game over. Well, probably not, but how many negative studies can you take?
Assuming the NIH/FDA findings are true, then the key question is not why most experiments were completely negative (there may many reasons why, for one thing they only tested XMRV), but why Lo didn’t find any XMRV amongst the positive CFS patients, and WPI didn’t find any MLV in their positive patient samples.
Two US cohorts of CFS patients with mutually exclusive presence of either XMRV or MLV, whereas the rest of the world finds nothing?? I don’t believe it. One would at least expect overlap.
My guess is that it must be something in the conditions used. Perhaps the set of primers.
As said, Lo used the same external primers as WPI, but varied the internal primers. Sometimes they used those of WPI (GAG-I-F/GAG-I-R ; F=forward, R=reverse) yielding a ~410 basepair product, sometimes their own primers (NP116/NP117), yielding a ~380 basepair product. In the Materials and Methods section Lo et al write “The NP116/NP117 was an in-house–designed primer set based on the highly conserved sequences found in different MLV-like viruses and XMRVs”.
In the supplement they are more specific:
…. (GAG-I-F/GAG-I-R (intended to be more XMRV specific) or the primer set NP116/NP117 (highly conserved sequences for XMRV and MLV).
Is it possible that the conditions that WPI used were not so suitable for finding MLV?
Lets look at Fig. S1 (partly depicted below), showing the multiple sequence alignment of 746 gag nucleotides (nt) amplified from 21 CFS patient samples (3 types) and one blood donor (BD22) [first 4 rows] and their comparison with known MLV (middle rows) and XMRV (last rows) sequences. There is nothing remarkable with the area of the reverse primer (not shown). The external forward primer (–>) fits all sequences (dots mean identical nucleotides). Just next to this primer are 15 nt deletions specific for XMRV (—-), but that isn’t hurdle for the external primers. The internal primers (–>) overlap, but the WPI-internal primer starts earlier, in the region with heterogeneity: here there are two mismatches between MLV- and XMRV-like viruses. In this region the CFS type MLV (nt 196) starts with TTTCA, whereas XMRV sequences all have TCTCG. And yes, the WPI-primers starts as follows: TCTCG. Thus there is a complete match with XMRV, but a 2 bp mismatch with MLV. Such a mismatch might easily explain why WPI (not using very optimal PCR conditions) didn’t find any low frequency MLV-sequences. The specific inner primer designed by the group of Lo and Alter, do fit both sequences, so differences in this region don’t explain the failure of Lo et al to detect XMRV. Perhaps MLV is more abundant and easier to detect?
But wait a minute. BD22, a variant detected in normal donor blood does have the XMRV variant sequence in this particular (very small) region. This sequence and the two other sequenced normal donor MLV variants differ form the patient variants, although -according to Lo- both patient and healthy donor variants differ more from XMRV then from each other (Figs 4 and 2s). Using the eyeball test I do see many similarities between XMRV and BD22 though (not only in the above region).
The media pay no attention to these differences between patient and healthy control viral sequences, and the different primer sets used. Did no one actually read the paper?
Whether theses differences are relevant, depends on whether identical conditions were used for each type of sample. It worries me that Lo says he sometimes uses the WPI inner primer sets and sometimes the other specific set. When is sometimes? It is striking that Fig 1 shows the results from CFS patients done with the specific primers and Fig 2 the results from normal donor blood done with the WPI-primers. Why? Is this the reason they picked up a sequence that fit the WPI-primers (starting with TCTCG)?
I don’t like it. I want to know how many times tested samples were positive or negative with either primer set. I not only want to see the PCR results of CFS-plasma (positive in half of the PBMC+ cases), but also of the control plasma. And I want a mix of the patient, normal samples, positive and negative controls on one gel. Everyone doing PCR knows that the signal can differ per PCR and per gel. Furthermore, the second PCR round gives way too much aspecific bands, whereas usually you get rid of those under optimal conditions.
Another confusing finding is a statement at the FDA site:
Additionally, the CDC laboratory provided 82 samples from their published negative study to FDA, who tested the samples blindly. Initial analysis shows that the FDA test results are generally consistent with CDC, with no XMRV-positive results in the CFS samples CDC provided (34 samples were tested, 31 were negative, 3 were indeterminate).
What does this mean? Which inner primers did the FDA use? With the WPI inner primers MLV sequences might just not be found (although there might be other reasons as well, as the less stringent patient criteria).
And what to think of the earlier WPI findings? They did find “XMRV” sequences while no one else did.
I have always been skeptic (see here and here), because:
- no mention of sensitivity in their paper
- No mention of a positive control. The negative controls were just vials without added DNA.
- No variation in the sequences detected, a statement that they retracted after the present NIH/FDA publication. What a coincidence.
- Although the PCR is near the detection limit, only first round products are shown. These are stronger then you would expect them to be after one round.
- The latter two points are suggestive of contamination. No extra test were undertaken to exclude this.
- Surprisingly in an open letters/news items (Feb 18), they disclose that culturing PBMC’s is necessary to obtain a positive signal. They refer to the original Science paper, but this paper doesn’t mention the need for culturing at all.
- In an other open letter* Annette Whittemore, director of the WPI,writes to Dr McClure, virologist of tone of the negative papers that WPI researchers had detected XMRV in patient samples from both Dr. Kerr’s and Dr. van Kuppeveld’s cohorts. So if we must believe Annette, the negative samples weren’t negative
- At this stage of controversy, the test is sold as “a reliable diagnostic tool“ by a firm with strong ties to WPI. In one such mail Mikovits says: “First of all the current diagnostic testing will define with essentially 100% accuracy! XMRV infected patients”.
Their PR machine, ever changing “findings” and anti-scientific attitude are worrying. Read more about at erv here.
What we can conclude from this all. I don’t know. I presume that WPI did find “something”, but weren’t cautious, critical and accurate enough in their drive to move forward (hence their often changing statements). I presume that the four negative findings relate to the nature of their samples or the use of the WPI inner primers or both. I assume that the NIH/CDC findings are real, although the actual positive rates might vary depending on conditions used (I would love to see all actual data).
Virologist “erv”is less positive, about the quality of the findings and their implications. In one of her comments (17) she responds:
No. An exogenous mouse ERV in humans makes no sense. But thats what their tree says. Mouse ERV is even more incredible than XMRV. Might be able to figure this out more if they upload their sequences to genbank. I realize they tried very hard not to contaminate their samples with mouse cells. That doesnt mean mouse DNA isnt in any of their store-bought reagents. There are H2O lanes in the mitochondral gels, but not the MLV gels (Fig 1, Fig 2). Why? Positive and negative controls go on every gel, end of story. First lesson every rotating student in our lab learns.
Finding mere virus-like sequences in CFS-patients is not enough. We need more data, more carefully gathered and presented. Not only in CFS patients and controls, but in cohorts of patients with different diseases and controls under controlled conditions. This will tell something about the specificity of the finding for CFS. We also need more information about XMRV infectivity and serology.
We also need to find out what being normal healthy and MLV+ means.
The research on XMRV/MLV seems to progress with one step forward, two steps back.
With the CFS patients, I truly hope that we are walking in the right direction.
Note
The title from this post was taken from: http://www.veteranstoday.com/2010/08/20/xmrv-renamed-to-hgrv/
References
- Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, Wang R, & Alter HJ (2010). Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors. Proceedings of the National Academy of Sciences of the United States of America PMID: 20798047
- Schekman R (2010). Patients, patience, and the publication process. Proceedings of the National Academy of Sciences of the United States of America PMID: 20798042
- Switzer WM, Jia H, Hohn O, Zheng H, Tang S, Shankar A, Bannert N, Simmons G, Hendry RM, Falkenberg VR, Reeves WC, & Heneine W (2010). Absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States. Retrovirology, 7 PMID: 20594299
- Erlwein, O., Kaye, S., McClure, M., Weber, J., Wills, G., Collier, D., Wessely, S., & Cleare, A. (2010). Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008519
- Groom, H., Boucherit, V., Makinson, K., Randal, E., Baptista, S., Hagan, S., Gow, J., Mattes, F., Breuer, J., Kerr, J., Stoye, J., & Bishop, K. (2010). Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome Retrovirology, 7 (1) DOI: 10.1186/1742-4690-7-10
- van Kuppeveld, F., Jong, A., Lanke, K., Verhaegh, G., Melchers, W., Swanink, C., Bleijenberg, G., Netea, M., Galama, J., & van der Meer, J. (2010). Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort BMJ, 340 (feb25 1) DOI: 10.1136/bmj.c1018
- Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, & Mikovits JA (2009). Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science (New York, N.Y.), 326 (5952), 585-9 PMID: 19815723
- Enserink M (2010). Chronic fatigue syndrome. New XMRV paper looks good, skeptics admit–yet doubts linger. Science (New York, N.Y.), 329 (5995) PMID: 20798285
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Related Articles
- Study Finds Retroviruses in Chronic Fatigue Sufferers (online.wsj.com)
- XMRV Virus Confirmed in CFS (psychologytoday.com)
- More Evidence Virus Plays Role in Chronic Fatigue Syndrome (nlm.nih.gov)
- Updates On XMRV Controversy (fightingfatigue.org)
- XMRV: Not in spooge (scienceblogs.com)
- MLV Joins XMRV As The Latest Unproven Cause of Chronic Fatigue Syndrome (psychologytoday.com)
- New Hope in Fatigue Fight (online.wsj.com)
- XMRV and chronic fatigue syndrome: Scientific Blue Balls (scienceblogs.com)
- Does X (the Virus, That Is) Mark the Spot in Chronic Fatigue Syndrome? (blogs.wsj.com)
- Presence of Murine Leukemia Virus Related Gene Sequences Found in CFS Patients (nlm.nih.gov)
- Betting on X – As in XMRV – With a Big-Ticket Research Center (blogs.wsj.com)
Laika's MedLibLog 
Interesting. Just wanted to point out a minor thing, that you use “CSF” instead of the correct “CFS” patients in a few paragraphs.
Thank you. It was far too late when I finished this post.
SEEING THE FOREST AS WELL AS THE TREES
Firstly, thank you Laika for another thoughtful and in-depth analysis of the XMRV and now MLV-related research. I genuinely look forward to the responses. I just wanted to add another piece of the puzzle, namely the big-picture view, with comments and pedigree from the FDA researchers themselves; as well as some more granularity on the Commentary on the FDA paper, for those who have not had a chance to read it.
As many of you may be aware, Dr Alter, co-author of the FDA paper, was co-discoverer of the Hepatitis C virus. This man has been around the virology block a few times, and indeed was a winner of the prestigious Lasker award, oft considered a quasi-criteria for a Nobel Prize (62 winners of the Lasker have gone on to win a Nobel Prize).
THE FDA’S PRESS TELEBRIEFING
In their telebriefing to the press, Drs Lo and Alter emphasized that the finding of MULTIPLE MLV sequences in patients with ME/CFS is entirely consistent with – and indeed typical of – retroviral infections in humans. Causality is yet to be determined, however research is already underway to do just that. And to use Dr Alter’s words, the FDA team found a “dramatic association” of MLV’s with ME/CFS, and in their words, their findings “basically confirm the findings of the Whittemore-Peterson group”. This isn’t WPI’s PR engine speaking – it’s Dr Alter and his team. I would encourage readers to visit transcripts of the FDA’s Press Telebriefing by the FDA team, including Dr Alter and Lo’s comments: http://www.wpinstitute.org/news/docs/FDAbriefing_082310.pdf and
http://www.facebook.com/notes.php?id=216740433250#!/note.php?note_id=432527206796
Some excerpts from the Press transcript:
DR ALTER: “So we found this (MLV-related viruses) in a very high % of the CFS patients that Dr Komaroff had sent to us – about 86%, and simultaneously found it in about 6.6 % of our healthy blood donors. “So there was a dramatic association with CFS with the syndrome of chronic fatigue.” But that’s all it is. An association and we have to emphasize as we did in the paper that we have not proven causality for this agent. There are many many more pieces that we have to fulfill. But we think basically it confirms the findings of the Whittemore Peterson group.”
“…It does at least confirm the findings of Whittemore Peterson. I think one wants to go back to their studies because they have had more time and more they have done extensive work… so it’s not just finding isolated viral sequences. But they have found gag, env sequences. They have been able to transmit this to an animal model (the macaque). They have seen antibody in the macaque and they have seen antibody in patients and they have seen the viral particles. SO THIS (Lombardi) STUDY IS MORE ADVANCED THAN OURS. BUT WITH THEM HAVING DONE THE GROUNDWORK, I THINK OUR STUDY IS HIGHLY CONFIRMATORY OF THEIR WORK and we are in the process of trying to culture virus from our patients and to find antibodies. We have already shown that some of our patients also have envelope sequences, and preliminary work appears that there is antibody present as well but this has not been confirmed.”
QUESTION: LAUREN NIERGAARD: ASSOCIATED PRESS
“I wanted to delve a little deeper into that as well. Does finding that its polytropic actually suggest a looser correlation with CFS than finding of Xenotropic would have?”
… “It would seem to suggest more of a process of chance than a firm association?”
DR LO. “No, actually this group of virus that we call polytropic MLV – this is actually MORE characteristic for the retrovirus infection instead of a single viral gene. They very quickly can evolve and have multiple different kind of a sequence found. In fact what we found is that actually gave us good confidence that this cannot be a laboratory artifact or PCR contamination product because the sequences they are so varied from one patient to another.”
LAUREN: “So that means it’s probably not lab contamination?”
DR LO. “Right, exactly. Normally the PCR lab contamination you more or less anticipate that all the sequences will be the same.”
LAUREN: “But what I was thinking of was not lab contamination, but rather you are finding essentially multiple different kinds of viruses within the same family.”
DR LO: “Right they are in the same family. They are compatible with the earlier finding of the XMRV. They are not identical, they are more diverse.”
LAUREN: “Is it usual that you would find an association with a single disease this kind of variety of viruses.”
DR LO: “YES, INDEED, THAT IS EXACTLY WHAT WE WOULD ANTICIPATE FOR RETROVIRUS INFECTION OVER TIME with the many different sequences there.”
DR ALTER: “Just on that last point, the retroviruses exist in big families of viruses. So Hep C is a very good example. Nobody is infected with one variant of HCV. It is a huge family of variants, HCV divided by genotypes and HIV divided by clades. And if you take any given patient the patient will have multiple variants in them. And different patients will have different variants in them. So it is very characteristic of these kind of RNA viruses.
DR MCCLUSKEY… “Just reiterating Dr Alter’s point. I THINK THAT IS THE ESSENCE OF THE CHARACTERISTICS OF THESE RETROVIRUSES. THAT THEIR POLYMERASE IS REALLY FAULTY AND CREATES LOTS OF MUTATIONS. SO YOU WOULD NEVER HAVE JUST ONE SINGLE SPECIES – causes multiple species…”
DR ALTER: “My thought came back. Since their original publication the WPI and NCI groups have now found that they too are finding greater variability among their patients, so it is not just XMRV even in the original cohort of patients.”
LAUREN: “So this is basically then saying that there is a stronger association than you had before because of that variability.”
DR LO: “I think we can say we found this kind of variability and that is actually more consistent with the natural form of a retrovirus infections in this group of patients.”
… and
DR ALTER: “Well, as with many things in science it is controversial when not everybody finds the same thing. Very good laboratories have come up with disparate results. And this is not totally explained. We think there are reasons for this. WE THINK IT IS IN THE PATIENT POPULATIONS RATHER THAN IN THE LABORATORY testing although the latter hasn’t been completely ruled out.
…. and
MICHELLE CORTEZ, BLOOMBERG NEWS: “If we have low levels of virus in the blood and yet we are seeing many variations on the sequence, can you speculate as far as what is going on. Is it some kind of.. is it mutating inside the patient? Or are we seeing there is quite a variety of virus out there and different people are being exposed to different kinds.”
DR LO: “Indeed for this type of virus this is quite characteristic. Once you infect in this case – earlier questions finding the virus gene sequence in the blood – that means this is an infection. This is a cell associated virus so of course if we truly find it in the patient blood we think that’s an infection going on. Although a virus titre or virus gene copy is very low. But finding when we sequence them and we finding a variation in the sequence and this is again like earlier description, THIS IS VERY CHARACTERISTIC FOR RETROVIRUS INFECTION AND WE DO ANTICIPATE THAT. In a way it was a little bit surprise when the WPI’s first publication in Science and they show it as a single kind of kind of a sequence. That is unusual for retrovirus, but now they are also stating they do see the variation of the sequence and also appear to be more closely related to polytropic related MLV’s.”
MICHELLE CORTEZ: “Would that be from mutations that are happening after the person is infected?”
DR LO: “Yes, that can be. Retroviruses use the RT, the reverse transcriptase and that is an enzyme that the fidelity is not very good so each time when they replicate they very easily introduce a different kind of a mutation. So after the infection certainly they can also start to accumulate this kind of mutation or changes.”
WHERE THE RUBBER HITS THE ROAD: RECOMMENDATION IN THE COMMENTARY
Finally, I would direct readers to the Commentary on the FDA paper, where in no uncertain terms the team advocates decisive action in the form of interventional clinical studies: http://www.pnas.org/content/early/2010/08/16/1007944107.full.pdf+html
“As we currently lack postulates to prove a causal association with a prevalent agent and a chronic disease with genetic predisposition, it would also be appropriate to conduct interventional studies. Indeed, the Helicobacter pylori hypothesis of peptic ulcer disease was only accepted after Barry Marshall showed that bacterial eradication with antibiotics cured peptic ulcer disease. STUDIES TO GAIN PROOF OF PRINCIPLE HAVE BEEN PERFORMED WITH ANTIVIRALS IN OTHER CHRONIC, IDIOPATHIC DISEASES LINKED TO RETROVIRAL INFECTION, SUCH AS PRIMARY BILIARY CIRRHOSIS ASSOCIATED WITH MOUSE MAMMARY TUMOR VIRUS, ANOTHER POSSIBLE MURINE ZOONOSIS. Trials using a combination of reverse transcriptase inhibitors led to significant improvements in clinical, histological, and biochemical outcomes in these patients, albeit with some evidence of viral resistance to therapy. SUCH STUDIES ARE NOW FEASIBLE FOR CFS, because reverse-transcriptase inhibitors, such as tenofovir and emtracitabine, and the integrase inhibitor raltegravir can inhibit XMRV.”
To sum up, some bona fide virology heavyweights have sounded off very enthusiastically on the FDA findings as being confirmatory of the Lombardi team’s work, and are indeed sticking their necks out for the patient and research communities by saying, “we’re ready” to start investigating proof of principle with ARV trials.
Suffice it to say that the patient community appreciates thoughtful discussion of the science. After all, this community has had experimental treatments (in the form of exercise therapy and mind control) fobbed off on us for decades – for what may turn out to be a retroviral disease. To put this in a context that many may understand, the “treatment” history for ME/CFS involving exercise as a cornerstone is akin to prescribing sugar therapy for a diabetic.
Thanks again for another thought-provoking blog, and I look forward to the continuing discussion.
Thanks so much @sciencebased for your kind and thoughtful response. I’m pleased that you appreciate my critical approach. And take it for what it is.
Many of the things you put forward are known to me. The point is that I rather rely on the publications themselves, then on what is said and by whom (nobel price winner or not). Patient often tend to do otherwise, because there is a lot of emotion involved and because it affects them directly.
For me it is “show me the data”. And yes I concentrate on the trees, the birds, the mold and the viruses because these make the “big forest” what it is. This is probably due to the fact that I have experienced once too often that small variations or simple things can make a huge difference, they can even determine whether an effect is positive or negative. And yes I’m intrigued by contradictions and things I can’t explain.
It is true that I don’t think positive of the WPI and Judy in particular. I wouldn’t call it bias, because I didn’t think this from the start. First I just found it very exciting. I felt sympathy for them, because I had been in a similar situation. I found a very rare sequence in normal individuals. No one believed me of course, and it took me 2 years to prove that I was right and it was no contamination.
You write in capitals that variation in the sequence IS VERY CHARACTERISTIC FOR RETROVIRUS INFECTION. That is true and that was my criticism against the science paper right from the start. One single XMRV sequence in every patient? Plus strong bands only after one round and only in samples of the Cleveland Clinic (at least in the Figure). Now they say they found more variation. That may be true, but what does this tell us about their original findings? Are they still true? Do all these patients still have that one strong XMRV-band? It wouldn’t surprise me if those bands were contaminations. Those things happen.
Then everyone suddenly states that culturing is needed for PCR to become positive. According to Judy it is in the Science paper, but it isn’t. So again what is beings said to the public and the patients is different from what is written in the article. Those discrepancies made me suspicious. Because if you can’t rely on what is written in a scientific paper, what can you rely on?
I also don’t like the attitude of WPI, but I must not reiterate myself. I have written about it in this and previous posts.
Here I also wrote about the shortcomings of the negative papers, not only with respect to the PCR technique but also with respect to the criteria of inclusion of CSF patients.
It is true that WPI did a lot more than PCR. It is easier to do a PCR than the other experiments, I suppose. That is a + for the WPI.
I’ve read about the idea to do a clinical trial.
Before this is done I really would like to see a confirmation that these MLV like sequences are more or less specific for CFS patients and not for other patients (in one experiment). MLV is already found in appr 7% of the normal individuals. Should we treat these healthy people too?
But in itself (as soon as it is more certain that these MLV ‘s are specific for CFS, such a study could solve a lot. Provided it is a controlled trial with a placebo and a retroviral treatment. (although I’m not sure whether there is a good placebo for this). I’m not a proponent of just try and see. Then we can’t proof anything. It is important that we do. For the patients.
Haven’t been a huge fan of the way WPI has dribbled out bits of new, unpublished info over time, either, but I personally found their actual science to be fairly solid for an early finding. Flawless? No, absolutely not. A good place from which to start looking and refining methods? Yeah, it seems to have been. I don’t think I’ve found any of the studies so far to be without problems, though, some more major than others.
Just as I personally try to avoid the “super-duper-respected guy found this, it must be TRUE!”, I also think there’s something to be said for avoiding “I don’t like how these scientists are doing X,Y,Z! Their findings must be questionable!”
Not that I think you’re doing that, exactly — point is, though, that one of my great annoyances with all of this great debate has been the injection of some kind of projected persona thing, on all sides. I don’t think McClure is an evil person out to prove that all CFS patients are loonies. I don’t think Mikovits is some subpar hack of a scientist trying to find the big bucks by telling patients what they want to hear. I don’t think Alter is… whatever he’ll be criticized for every alternate Friday until the next person publishes something on the topic. If any of those things were true, it shouldn’t really matter anyway, unless one thinks they’re actually lying in their published results.
Early findings in any science should be met with skepticism, until research has responded to reasonable criticisms. Skepticism isn’t the same as flat doubt, but this whole process relies on people poking holes wherever they see possible problems. Still, this is a much better lead than anybody doing CFS related anything has had in ages, just by nature of it pointing to _something_ that may be related and across multiple studies. The agony of people who have this thing includes the awful symptoms, and the lack of any solid information whatsoever, and the historical attitude of many doctors, so they are absolutely going to continue hanging on every word of it, as well they should. Of course, that also means that scientists’ frequently poor communication and media-wrangling skills will absolutely be played out in public here.
In some ways, if this stuff pans out over time, this is a more interesting finding than if they’d simply also found some XMRV in 2/3 of the patients. I love the weird findings — they’re to be met critically, of course, but it’s simultaneously true that the “WTF!” results are often the ones with potential to lead to really major new insight.
An excellent post.
So speaking as a non-virologist, am I right in thinking that the key question now is – why did the WPI find XMRV with their, XMRV-specific primers, when according to Lo & Alter, there is no such DNA present in either patient or control samples (although there is lots of related MLV DNA)?
I’m no virologist either 😉 But I’ve worked on herpes viruses and have done a lot of PCR-ing. That is why I know the pitfalls so well.
Yes, basically that is the major issue. And also the other way around. By using “XMRV”-specific primers Lombardi et al might have missed the other MLV-like viruses (in their Science paper).
Those primers (and the conditions used for PCR) define what is amplified and how specific the PCR is. A mismatch might mean it is easier to amplify XMRV then it is to amplify MLV.
You can also see that there are a lot of “competing sequences” expecially after the first round. These competing sequences disappear if a positive signal wins. If you look at the gels you see almost no aspecific bands in the plasma (no competition), and less aspecific bands after a positive signal in PBMC. (alas copyright doesn’t allow me to show their figures)
I was very excited when I found those differences, because it might (I emphasize might) explain the differences between the findings of the two groups. I wonder why it isn’t more stressed in the paper. The authors mention it twice in the materials and method section, i.e. (GAG-I-F/GAG-I-R (intended to be more XMRV specific) or the primer set NP116/NP117 (highly conserved sequences for XMRV and MLV).
Since their conserved set should be able to pick up both XMRV and CFS-types MLV, as they call it, it is surprising that they didn’t find XMRV. I doesn’t seem likely that the WPI-CFS population contains only XMRV (with exactly the same sequence in all samples) and the Lo/Alter population contains only CFS-type MLV and no XMRV whatsoever.
So you might look at the results as a half glass empty or a half glass full.
Full: the Lo/Alter study did confirm the WPI findings with respect to the presence of retroviruses in CFS-patients.
Empty: the two studies find entirely different retrovirus-types. As ERV explains XMRV is very different from the CFS-type MLV’s
There are also other striking findings that are almost ignored. There are 3 CFS-types in the CFS patients. Most of the patients (18) have type 1, 2 have type 2 and one has type 3. These are just marginally different in their gag-sequence. What I really find interesting is that the gag-sequences found in the normal MLV+ controls are of another type and have more differences in their sequences compared to the CFS-MLV types. By the way, their “mutations” are often identical to those in the XMRV gag sequences.
Could the demonstration of ‘CFS-MLV-types and ‘healthy-control-MLV-types’ be relevant? Or is it an artifact? For instance dependent on the primers used. In their figures Lo et al state that they used specific primers to test the control samples (which picks up more XMRV-like sequences in this gag-region), whereas they used conservative primers for their patient series. I don’t say they always did this, but I find it strange they don’t show the results with one and the same primer. Or two figures, one with each primer.)
That is one of the downsides of researchers just showing their best pictures or gels. I really would like to see them all, with positives and negatives next to each other.
Neuroskeptic, no, Lo and Alter did not say there is no XMRV in their samples, it is just not what they found. This whole area is so new, MLV’s (and XMRV is an MLV) are not tightly defined yet. Alter clearly said his study supports the findings of the WPI. The WPI have, since publishing, found other MLV’s in their samples.
As a non-virologist myself, I had it explained thus: Dog is to terrier as MLV is to XMRV. Hope this helps.
@jace The whole point is: we are (I am) not speaking about what they are saying, but what there data are showing, thus what they are finding. They didn’t find XMRV, that’s what we are talking about. So we are talking about the same.
And I’m not talking about what the WPI is saying they are finding now, but what their data published in Science say they found then. We are only able to interpret their new findings once they have been published. Otherwise we are only speculating. Furthermore WPI is always saying different things then they have published, so that makes me cautious.
SB– Thats nice that Very Important People think this is ‘good science’. Very Important People are also baffled as to why other Very Important People think this is ‘good science’. Science has relatively little to do with Very Important People.
jace– “Dog is to terrier as MLV is to XMRV.” Kind of.
Dog is to terrier/bulldog/labrador as MLV is to MPMV, PMV, and XMV.
Terrier/bulldog/labrador:Mexican wolf as MPMV/PMV/XMV:XMRV.
They are all superficially ‘dogs’, or ‘MLV’, but you dont confuse a wolf for a terrier/bulldog/labrador. XMRV does not look like MPMV, genetically. Fundamentally it has LTRs, a gag, pol, and env, but XMRVs LTRs are 200 nt short. Its gag has a distinctive deletion. Its env has a different tropism. You just cant confuse these things, or use the words interchangeably.
Also, in their phylogenetic tree, they compared the found sequences to endogenous retroviruses. ERVs are frozen in time. They exchange their high mutation rates for the more conserved mutation rates of their host. Finding an endogenous sequence with the kind of similarity Lo et al found in an ‘exogenous’ virus (some not branching at all) is baffling.
laika– *nod* Very nice!
They just uploaded a handful of their sequences to Genbank for outside analysis. 1) There is no sequence diversity in the CFS samples. Two different nucleotides out of 700 nucleotides is not ‘diversity’. 2) The sequences are plainly mouse ERVs when you BLAST them.
If *I* did this experiment, and I got these results, I would think I contaminated my samples with mouse genomic DNA. I would not be convinced of this data if it were *my* data.
What would have resolved this, is if they did what the independent reviewer asked, and mapped integration sites. But they didnt want to do that.
:-\
ERV: They tested for mouse mitochondrial DNA, and found none, but I take it you’re arguing that they should have tested for mouse genomic DNA too?
We’ve mapped the mouse genome and the human genome, right, so it can’t be that hard…
Mapping integration sites is obnoxious and time consuming, but it is not hard. Single-genome PCR. You know what youre looking for– point the primers out instead of in. BLAST the resulting sequence.
‘What would have resolved this, is if they did what the independent reviewer asked, and mapped integration sites. But they didnt want to do that.’ I think that is what they are doing now.
After they told the independent reviewer ‘No, I dont want to for this paper’ and ran around the press junket saying they ‘found something!’.
There is no way I would have published with the data they have in that paper, especially seeing the sequences now. They are going to have to retract if there is contamination and these are not real ‘mouse-virus-in-human’ integration sites.
Id rather wait six months to publish a paper than rush to publish and end up retracting. Id rather cut off my arms than have to retract.
I ‘m not sure @erv. There was a lot of pressure on them to show what they had found till now. Everyone was speculating. So wouldn’t it be better to show what they got? They agreed with the independent reviewer, that it would be an essential thing to do, but said it would take too much time right now. They probably are working on it now.
If that was a sensible thing to do the future will tell.
Unlike you I’m not convinced it is contamination. All the sequences differ from the controls they used, and they find variation of sequences within their patient population. Furthermore some of the sequences were still present in fresh blood samples and seem to have evolved (somewhat) over time. The sequences in control samples are also different.
On the other hand, the results are over-interpreted by many. And especially by patients. It is so important that you don’t create false expectations.
If I ‘cure AIDS’ tomorrow, and there is a lot of social pressure for me to publish a paper I am not totally comfortable with, scientifically, Im not publishing it.
The angry mobs of people INSISTING the paper be published ASAP arent the ones that will have ‘RETRACTED PAPER–>SHIT SCIENTIST’ tattooed in red ink on their foreheads.
Im also not impressed with the fact this lab did not think to do this on their own, without nudging from the independent reviewer. Furthermore, they have uploaded a grand total of three ~700 bp sequences from ‘CFS patients’, and the sequences theyve uploaded are virtually identical, save a nucleotide here and there. I dont care if they say they ‘found diversity!’, there aint no diversity in the sequences theyve uploaded so far.
[…] controversy with another comprehensive treatment prompted by the newest paper in the field – Does the NHI/FDA Paper Confirm XMRV in CFS? Well, Ditch the MR and Scratch the X… and… you’ve … And Abbie at ERV covers the same paper without mincing her words – ouch! – in XMRV and […]
Erv give it up. Let’s make a rule before attacking the research of highly respected scientists Dr.Alter, Dr. Lo, Dr. Komaroff, Dr. Goff, Dr. Coffin, Dr. Silverman, Dr. Ruscetti etc. each of whom have written more peer review research articles exceeding your own age and have distinguished themselves in their fields of study, have received numerous medals and awards for meritorious achievements in scientific breakthroughs, list your accomplishments to date.
Erv….PhD =0
Erv….peer review articles to date =0
Erv….scientific breakthrough research=0
Erv….published scientific journal articles to date hmm, does seed magazine count? But then grad students do all the work, write all the research papers and the PhD’s, they just sign their name to the paper and get all the credit..wink…wink.
Who is Erv but a struggling grad student attempting to work her way through some no name grad school in the middle of hick town OK. The only person that will have ‘Shit Scientist’ tattooed on their head is you Abbie and don’t worry you will never cure Aids. So put away your chemistry set that your parents bought you when you were a little girl and please for the sake of humanity seek therapy…I know you might lose your groupies but it will do wonders for your egomaniacal attitude.
@spit is right. It is not about “personas” (sorry @spit, I wrote a large comment yesterday eve, but pressed a button -accept your comment again- and it was gone. was too tired to rewrite it).
Erv critics not the people in the first place, but their work. Whether you have published yourself or not is not really relevant, as long as it matters what you say. Thinking in the same line: patients that haven’t studied can’t evaluate what has been published, because they are no scientific researchers. Rubbish. The other way around: being a well known researcher doesn’t give you an aura. So people lets talk about WHAT has being said here. It really frustrates me that the discussions are again about people (and since the comments are about people I cannot avoid talking about it too). Few people go into what I said about the studies themselves. For instance about the new aspect: the use of different primers in both studies.
Erv said some sensible things, i.e. the explanation of how XMRV relates to MLV, about the need to 100% exclude contamination, about the very small variations in the MLV’s. Please listen more to the things she says then the tone.
And by the way. As a researcher I published about 20 papers, many about PCR. One of my co-authors is van Ommen (HUGO-project). That means that I never ever could have been wrong and I never ever had contamination in my PCR.
I’m sorry to say that isn’t true. But, as erv advises, we just went on researching and excluding artifacts till we were sure we were right. For my most important paper, Oncogene asked us to do one other experiment to exclude contamination. It took me another two years. And I’m happy we did it that way.
Bleh, no worries. I hate the lost comment thing. I have an amazing ability to do that, too. Happens every time I write something really brilliant, of course. 😀
Taking my own advice and avoiding any comment, positive or negative, on erv and her tone, I will say that I’m largely interested in, though not immediately convinced one way or the other by, arguments that would point to contamination going forward on this one. Mostly because when I first read the paper, I thought, good, they did a whole lot to show that this wasn’t probably contamination. I’m not anything like a virologist, though, so I know damn well there are many finer points that I’m not going to see right off.
I’ve been very wait-and-see with this stuff, honestly, and I think the only thing that really bugs me about the criticisms I’ve read of these studies is that they aren’t always really outlining the actual problems. “It doesn’t make any sense!” is not really an argument or criticism, and part of why I love to read science blogs is to see what people actually in the field in question think of the data, the potential pitfalls, whever. The studies that have been done, both positive and negative results, have all seemed to have some problems to me, but again, this is not my field — dammit, Jim, I’m an ecologist, not a retrovirologist! I’d love to see people who work on this stuff really tear into the research, in a critical-but-not-pissy way. Many kudos and thanks to you for doing a lot of that here, it’s a good read.
Rigor is important to me no matter what the results — not because of reputation stuff, or because I expect it all to be perfect, but because the better the study, the easier it is to figure out how to look at the questions or problems it brings up. Not finding something means… you didn’t find it. That could be that it isn’t there, and it also could be that we don’t know what this thing is doing and aren’t very good at looking for it yet — those questions are much easier to figure out if the researchers are really self-critical and make every effort to cross their i’s and dot their t’s.
On the PCR — I haven’t delved into the details of these primer sets the way you have, but had been wondering about the primer differences in all of these studies generally. If WPI used a primer set that couldn’t catch small variation from (or within?) XMRV, somebody should really get on clarifying that with their samples with different primers (via publication, please!). Their choice of internal primer may well have been intentionally made to exclude MLV type stuff, I’d guess, since they were looking pretty specifically for XMRV and were likely trying to get around contamination issues just as hard as anybody else.
But like you, I can’t imagine why this study wouldn’t have found any XMRV whatsoever, at least based on the primers. It’s hard to even come up with guesses, unless I go to sample handling or some small regional variation, dunno; arguments about patient selection don’t possibly explain the degree of discrepancy in any of these studies, as far as I’m concerned. I mean, what, the CDC didn’t even get ONE person with this thing using their selection criteria, when apparently 4 – 7 % of even the control came out positive? No. There’s something else here.
Figures that a controversial set of research on a controversial disease would just have to go and involve something in a group that contaminates the crap out of everything, doesn’t it?
Those are good points you are making. To a certain extent patient selection and sample handling might make a difference (see below). But they cannot explain the differences in results between the Lombardi and the Lo paper. I really have to grin when people say that Lo found a XMRV variant. That simply ain’t true.
I think it was like this. WPI had an idea that there might be XMRV in CFS-patients. The rationale was that both XMRV-positive prostate cancer (XMRV had originally be found here) and CFS, have been linked to alterations in the antiviral enzyme RNase L. So considering the hypothesis it is only logic that they used XMRV-specific primers. The conditions were not documented very well and the PCR products obtained were very homogeneous. Strong bands were obtained after one PCR round. However later they said that culturing was needed to get a signal even after 2 rounds (which was not mentioned in the Science paper). Homogeneous sequences, and strong bands after one round (which isn’t reproducable later on) might indicate contamination.
The 2nd to the 5th study had less rigid criteria for including CFS patients, but here you are right, you would at least expect some people to be positive (10-30% at least: controls and some ‘real’ CFS patients). Provided the PCR was ok. The 2nd study (Erlwein 2010, UK) however used other primers, didn’t purify the blood cells (dilution signal and possible inhibition by granulocytes). Their gels look bad with weak signals. The 3rd (UK) and the 4th study (Netherlands) had excellent PCR’s and used the more sensitive real time PCR. The sensitivity was great, but the fourth study was very small (with no strict criteria). But these patients were not from the US, so that might be another reason why the PCR’s were negative. This reason could be dismissed, because the WPI stated that they had found positives inone of the UK studies and the Dutch study. Now this is overlooked by many, but it would mean –it is just logical thinking- that there was either no wrong classification (but poor PCR-but this isn’t true) or that WPI contaminated those samples. There is no other explanation. However this is all NOT published, just a letter by WPI.
I didn’t look at the CDC study yet, because it was the 4th negative study in succession and it got boring.
Till the last study I thought that the type of primer wouldn’t matter as long as the sensitivity or specificity for XMRV was all right. And this is true as XMRV is concerned. But not if you want to find other types of MLVs. Why Lo et al also tried other primers I don’t know. Perhaps when they didn’t find any XMRV? And because the XMRV primers weren’t optimal to find MLV (perhaps they found weak bands with it)? It is a pity they don’t document this well. It is also odd that the figure shows controls sequences amplified with the XMRV-specific primers and CFS-material amplified with the general primers. Especially because the MLV from the normal controls have another configuration. Is this real or caused by the use of another primer (you only see what you amplify)?
Unlike erv I don’t see evidence for contamination. Imo the sequences differ enough from the positive controls that contamination (by PCR-products) is unlikely. The results also seem consistent. But whether small heterogeneity within patient samples is a likely phenomenon, is another point.
Thus yes, I can understand why WPI couln’t find the CFS-MLV’s with their primers, but I can’t understand why Lo et al wouldn’t pick up XMRV (especially because almost each and every WPI-patient has this variant).
Thanks, it is nice to have this discussion with you.
Good comment, and it’s been really nice to see actual discussion of the science — hugely heartening, really. This whole subject has been driving me nuts for a while; I don’t even mind vitriol, generally (lord, it’s the internet), as long as the actual flaws or merits of the science are also being discussed at all thoughtfully.
I haven’t known at all what to make of WPI’s unpublished results, except that I don’t try to parse them too much, and it’s really annoying to me, too. I do understand some of it, from a researchers-are-still-people point of view — they’ve got patients absolutely clamoring for information, and I’m hugely sympathetic to the need for answers as quickly as possible. I’m sick, too, not (so far as I know) with CFS, but with likely narcolepsy, which is another medical treatment and research quagmire. The idea of waiting potentially several years for anything that might suggest better help has this way of sounding like forever when everyday is full of more suckitude. I know well how research works, and I _still_ get really impatient with it, because dammit — this sucks, my life feels nearly totally hijacked, and the idea of that just continuing indefinitely is claw-your-eyeballs-out maddening.
Still, it’s impossible for anybody to really sink into stuff that comes out as press releases instead of data, and while I understand that they’re also feeling responsible to patients with the disease — their overall mission is mixed, and I don’t think that’s a bad thing, in the grand scheme — there does come a point where you’ve got to _formalize_ the results you’re talking about and subject them to detailed peer review, if you want anybody to actually take them seriously. Otherwise, it’s all grain-of-salt stuff; I refuse to take it as more than hearsay, and so should everybody else, until they give us data and so forth.
And I do agree that the negative studies _after_ the Erlwein study (which I found very thin) looked pretty solid too, overall, at the very least on the PCR itself. So that leaves major (_really_ major, no overlap whatsoever) population differences, contamination in the original study, or some other totally unknown weirdness (earlier handling/purification/sampling problems?), maybe.
I think if this new study had also been negative, I’d have been pretty ready to accept contamination as a likelihood, unless there was a release of amazing new data. At some point, you’ve got to put up or shut up, and new published data with a detailed methodology would, not to endlessly repeat the point, be very, very helpful from WPI.
Of course, a positive result would have been pretty solidly pointing toward mistakes somewhere in something we don’t understand, some sampling that went awry or some weird behavior of something. Because yeah, I think it’s fairly hard to criticize the PCR itself on several of these, but I’m also pretty open to us having a lot to learn about XMRV still, and now potentially also some closely related MLV variants.
Instead, we got neither positive nor negative, exactly — we got weird. But weird in a way that I think tells us there’s very likely _something_ at play here, MLV/XMRV-wise.
I’d love to know what prompted the broadening out of primers to include MLV, too — I also suspect they didn’t find XMRV but maybe got some other weak signals, or maybe were looking for small variations in loci for the WPI primers to rule out problems there, but there are a number of things I’d love to have seen them clarify more. The more detail, the better, especially in research bound to be argued over. It’s always the tiny detail that makes or breaks research, and it drives me nuts when results are published without enough of it.
The rumor mill points to more studies published very soon on the topic; I should take bets on whether they make things more confusing, or less so. If there is another result that’s all yes-no-sorta like this, I honestly would just flat support carefully designed research on the effects of ARV treatment in a carefully selected patient population. It would be a little fraught with peril, too, but would at least give another direction to work from while the “which, what, how?” level of sticky contamination questions, viral phylogeny, and physiology work out.
Eco. All the ad hominems in the world wont make the sequences Lo et al submitted diverse. It wont justify Mikovits accusing other scientists of fraud and bribery, or her support of anti-vaccination.
Sorry for Ecos comment, laika. I has a fan club 😀
We’ve mapped the steal genome and the frail genome, change, so it can’t be that hard…
I has a fan club too. Erv, I support your last comments about Judy with one exception. Will you stop projecting yourself on to someone else. You are unconsciously denying your own attributes, thoughts, and emotions by projecting them onto another person…classical Freudian psychology. Make an appointment with Wessely. I’m sure some CBT will help.
It’s a shame the negative studies tested patients who didn’t have the disease. It’s good way to make the disease disappear. Significant number of patients in the CDC study or their Publisher’s Clearing House cohorts claim CFS but never went to a doctor for treatment, never received a prescription for their condition as alluded by Dr. Alter in follow up conversations.
Social Policy in the UK and Netherlands will never admit that this disease has a pathogenic origin. Unum and all the other medical & disability insurance companies along with the psycho-babble crowd will prevent it. Too much funding flowing toward Wessely for his pet projects.
@Eco The fact that the FDA/NIH/Harvard team didn’t find any traces of XMRV kind of exonerates the negative studies of the accusation of bad science.
I am an ME/CFS patient myself, and had the honor of being quoted as a critical CFS patient in the article, but I am not betting any money on XMRV or MLV yet. Depending on the presentations at the NIH workshop and future publications, I might be tempted to give the new serology test a try, more out of curiosity than as THE answer.
A lot of questions can be asked on how ME/CFS has been treated these past decades, and I hope one day they will be answered, but I find conspiracy theories fatiguing (pun intended). There are precedents: asthma, MS, … have all been considered between the ears once.
I’ve read some new posts by
you, Johan, and yes you’re “critical”. Mind you critical in a positive way. You just don’t automatically accept everything that has been written about CFS.
Conspiracy theories are fatiguing, even for me.
I don’t believe in conspiracies. I do believe that science is not always without mistakes. And that there can be conflicts of interests and other forms of bias.
I don’t believe in a psychogenic cause of CFS either. At the most certain therapies (BHT) might help (a bit) to coop with the disease / the extreme tiredness.
But BHT doesn’t take away the cause.
A viral cause seems more likely.
Evidence about BHT:
http://www.crd.york.ac.uk/CRDWeb/ShowRecord.asp?ID=12008103463
http://clinicalevidence.bmj.com/ceweb/conditions/msd/1101/1101.jsp
@eco Just want to repeat my previous remark that Annette Whittemore wrote the following in an open letter to McClure (virologist who did the PCR for the 2nd study, co-authored by Wessely, and author of an editorial in the BMJ responding on the negative Dutch trial)
“We would also like to report that WPI researchers have previously detected XMRV in patient samples from both Dr. Kerr’s and Dr. van Kuppeveld’s cohorts prior to the completion of their own studies, as they requested.
Now if all of these patients are not CSF-patients -as you state=, how come that WPI can detect XMRV in them (as they claim)?
No and it is not the failing PCR, because in both cases these are extremely sensitive (for XMRV that is).
Open letter:
Click to access DearDrMcClureaw4.pdf
Great post, Laika. The technicalities behind the issue are complicated enough, apparently even for virologists and ‘PCR-logists’ themselves!
Anyway, I think you and ERV raise some very valid points about the specific methods used and the results reported on the mentioned papers. And I stress the “reported-on-the-mentioned-papers” part. Press releases and TV interviews are definitely not the right places to announce new results before reporting them in full in a peer-reviewed journal…
I agree that a great part of the problem stems from the seemingly incomplete descriptions of basic experimental details and results (origin and handling of samples, PCR reactions, negative/positive controls, sequences?). Ideally, all those details should be fully and clearly described in the original papers. Especially if the research tries to shed light into an already confusing field, and might potentially affect the lives of so many people.
Without all the relevant data, we could be discussing for ages and getting nowhere. I very much hope that new, more complete reports will be published soon. With plenty of data and experimental details!
And, yes, it’s a shame that some media and some people keep focusing on “who said what”… That’s not the point at all.
Well said César. I have nothing to add. Thanks.
Thanks all for this great discussion.
Good tip @science_based. Hope it is eastern time in the US. Otherwise it will be too late for Europe to follow (and even then).
Meanwhile you may be interested to know that a second WPI paper has been recently published.
Detection of an infectious retrovirus, XMRV, in blood cells of patients
with chronic fatigue syndrome
Judy A. Mikovits,1,* Vincent C. Lombardi,1 Max A. Pfost,1 Kathryn S. Hagen1 and Francis W. Ruscetti2
1Whittemore Peterson Institute; Reno, NV USA; 2Laboratory of Experimental Immunology; Cancer and Inflammation Program; National Cancer Institute-
Frederick; Frederick USA
http://www.landesbioscience.com/journals/virulence/article/MikovitisVIRU1-5.pdf (freely available).
I didn’t have the time to read it from A to Z. It is published in the new, and unknown, journal “virulence” as an addendum to the science paper (!)
I have never seen such a thing (publishing an addendum in another journal).
In the abstract it says:
In this addenda, we further detail 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.
Good that the data are now published. And if I hadn’t seen the bands in the Science paper it would have made perfect sense. BUT
the PCR from unstimulated PBMC gave very strong bands after a single round. So what happened in the few months thereafter? Why wasn’t this immediately published in the Science paper, even as an addendum perhaps? PCR got weak?
I will dive deeper into this soon.
Laika, thanks again for your continuing thoughtful analysis and temperate comments. Your readers may be interested to tune in to the Question and Answer session for the 1st International Workshop on XMRV, hosted by the NIH, which will be held this coming week in Bethesda.
The Q&A Wrap-up Session will be aired Wednesday, September 08, 2010, 5:15:00 PM, and you can view the event online at http://videocast.nih.gov while the event is live.
Recognizing that academic pedigrees don’t guarantee scientific accuracy, many of the leading XMRV/MLV researchers/policy makers will be there, including Coffin, Silverman, Klein, Singh, Mikovits, the Ruscettis, LeGrice, Holmberg, (and I believe Lo/Alter), etc. Needless to say, many of us will be glued to our laptops.
Keep up the great work, Laika.
OK, no need to say it but excellent detailed post! I have some comments, none with real depth because I have not read all the paper or followed up on the sequence analysis etc. It’s a strange story so far – you would think that if it’s there you find it, if it’s not, you don’t – until you think about the complexity, as you describe very well above. It reminds me a bit of the helicobacter / ulcer story which was pretty controversial in the beginning but ended up with Nobel prizes (and a proper cure) – it would be good if this turned out to be true.
But here is an issue, the murkiness of the situation as you describe it is very worrisome because the potential rewards and prizes are so great. What troubles me most of all though is not the squabbles, the truth will come out sometime, but the fact that there is already a commercial test that WPI are making money out of. That stinks. It’s a) far too early for a patient test b) what is the intervention if the test is positive? retrovirals? It is simply unethical. There has been a lot of debate recently about genetic testing and the dangers of DTC so the FDA looks like it will be taking some action. I argued elsewhere that non-DTC (i.e via MD tests) are potentially MORE harmful and need to be regulated more thoroughly than DTC (http://bit.ly/agjs9v plse excuse self pimp!). This XMRV test (ha! which will “use methods that will detect all known human MLV viruses” http://www.vipdx.com/) is an example that supports that argument and it also supports the case that these sort of LDT tests should be regulated. This is pure shameless exploitation of a very vulnerable population that is desperately searching for some answers and treatments – the CFS patients deserve better than this.
It’s an interesting point, and one that I know has generated heaps of heated discussion all over the place. Here’s my personal take, for what it’s worth, because while I don’t think it’s good, I also don’t find it hugely surprising, and my view on the ethics of it is complicated, so bear with me a little on the length.
There’s a very real perception out there — and I don’t think it’s coming out of nowhere, I think there’s something at core of it that’s very much real, based on my personal experience — that there’s this nexus of insurance company meddling and lack of research funding and overworked-underconcerned doctors that makes it very hard for patients with difficult diseases or symptoms to feel they have anybody really working hard to help them find answers. I _don’t_ think it’s that most patients expect answers to be made up when there are none, I think that’s largely a false image. But I _do_ think that most patients with hard to pin down symptoms feel very quickly like they’re being cast aside, losing access to possible testing or at least symptomatic treatments, fighting like mad to just stay on the radar and not be shunted off when the basic bloodwork comes back looking fine.
I think that’s also exactly why you’ve got a rise in independent labs doing this stuff, why there’s such a large market for it. I think there are real, true, valid reasons patients feel that way and go looking for other possibilities, some good, some less so.
AFTER explaining my family history that _should_ point to particular tests, I have still had to plead for them, argue for them, wrangle with specialists. I have explained to doctor X that some days I can hardly walk around because my legs feel wobbly, only to have doctor X tell me to walk more often so I’ll lose the weight I’ve gained since getting so sick. I have had doctor Y tell me things about condition Z that he would have known were flat out false — not controversial, just false — if he’d even just read the WebMD article about it, much less flipped through any remotely recent journals in his own specialty. It’s been hard to feel confident that I’m getting the tests that may help, it’s been hard to feel confident that anybody has been actually considering any of it at all, and it’s really hard to fight all these battles when you’re sick.
I think this is the part that’s so maddening to me about some of us being so quick to call out the people involved for ethical considerations, while simultaneously refusing to discuss anything about the people who have the disease. You can’t understand why those things are happening at all, if you don’t want to understand anything about patient experience. If you want to stick to science, stick to science; if you want to comment on the ethics outside of the published research, that’s a messy, messy realm right now, and I’ve come to the conclusion that there’s absolutely no way I can confidently condemn or happily approve of almost anything going on in it.
I don’t like that this test was made commercially available so quickly, I don’t like that it’s at all connected to WPI (but that’s likely part of their dual mission), I think it’s a mistake to put any test out into the world until it’s clear how results should be interpreted, until the sensitivity and specificity are very well understood, so forth. I think what all of this research squabble points to is that we don’t know what we’re doing with this thing yet, even just in terms of consistently finding it at all. Waiting another six months or year to make sure the testing itself is good is hardly a huge wait, especially given that we still don’t know whether this causes anything or whether it even can be found with any reliability. Shit, as the NIH study makes clear, we’re not even entirely sure what we’re trying to find, yet.
Simultaneously, I don’t think it’s that patients are being somehow “duped” into getting tested for something too early. I think they’re damn sick of feeling like their concerns aren’t being seriously explored generally, and if there’s a potential for a new thing to explore, it’s human and right to want to know more about this thing that may potentially be turning one’s life upside-down. Most of the patient discussions I’ve seen on this stuff — both on the testing and the potential for ARV treatments — have been pretty careful to point out that this is all very new, that methods aren’t worked out, that findings are still weird, that we don’t know whether this is causal or related to much of anything, and that antiretrovirals are very nasty things, not something you really just want to fiddle with willy-nilly. People aren’t stupid, by and large; if you look for problematic stuff, you’ll find it, but on the whole I think people do understand that this is not untangled yet, that there’s a long way to go.
Imagine, for a second, that you feel like utter crap every day, that you feel isolated (chronic illness is extremely isolating in our culture, even when people don’t treat you like you’re crazy), that you feel unable to function sometimes on basic levels, and that your most major daily concern is your health — not because you’re “obsessed”, but because you can’t realistically forget that you’re sick for a single second. Can you see how people’s threshold for trying something new might be altered from the “we don’t know enough!” of the healthy population?
Physicians are the gatekeepers in our system, and that’s good — I have seen what some of my (hippie, whole-earthy) friends have done on their own with some medical issues, and it ain’t cool sometimes — but being the gatekeepers, IMO, also means extra responsibility to work thoughtfully with people to find the best things to try. “Do no harm” is a complicated statement, to me, because I’ve also come to realize that a combination of doing nothing and not allowing me to do for myself is sometimes incredibly harmful. Until a lot more people understand that, there’s going to be clamoring for this stuff, and I can’t wholeheartedly condemn it when I look at the way what we’re doing lets so many people suffer without help for so long. “Maybe this will be helpful to know, maybe not” is a hell of a lot better than any of these folks have had from most of the medical world for a long, long time.
@keith It reminded me of the Helicobacter (and the HIV) story as well. This story might end with Nobel Prices as well, but it might also end (as someone dm-ed me privately) as a Wakefield-story. As you may remember Wakefield claimed that vaccination lead to autism. But later it appeared that most measles viruses PCR’s had been negative and all positives had been contaminations. The false positives derived from plasmids with measles sequences in them (the positive controls). No, as said earlier, contamination is not an improbable thing at all.
Furthermore, like WPI, there appeared to be a conflict of interest (Wakefield being paid by a legal firm representing parents against a drug company).
Another parallel: Judy went to an autism meeting recently to tell about XMRV in autism. In the early days (Oct. 2009) she told on the Nevada television that she found XMRV in 40% (!) of the autism patients and here she also speculated how vaccines would lead to autism….
(This claim alone underscores that it is so important to test whether XMRV is specific for CSF)
Yes it is really really worrisome that Vip Dx sells a commercial tests (licensed by WPI, as if that is a proof of its robustness) that has not been proven to be diagnostic. I’m glad that you extensively explain it in another comment, because it is hard for people to understand why this is not the way to go about.
(I have written about vipdx in a previous post)
How can it be, that a XMRV test is licensed as soon as the XMRV test is published, and a XMRV ànd MLV serology test as soon as the Lo/Alter paper about involvement of MLV comes out.
Yes, antivirals are the next step. People are already taken them, also in the Netherlands/Belgium.
The Belgian Prof Kenny de Meirleir has just announced that he also detects XMRV in European people. And you know what? The lab which does this testing is RedLabs (Vip DX was the USA branch of RedLabs).
Many patients have undergone a XMRV test. Some take antivirals. Some are confused, because not as many people mention their test to be positive as one would expect a priori. Some wonder, should I be tested for MLV’s too? Should my immunological signature be tested by de Meirleir’s (who claims CFS patients have a HIV -like signature, which also occurs in ….. autism for instance), others wonder what it means if they tested negative for XMRV.
http://www.huffingtonpost.com/david-kirby/is-autism-associated-with_b_316986.html?view=print
http://leftbrainrightbrain.co.uk/2007/06/andrew-wakefield-and-the-death-of-the-mmr-debacle/
Laikas, that’s a very good point about wakefield/autism etc. Lancet paper on a few samples, prestigious institution, press released, etc… I sincerely hope that this virus stuff doesn’t lead to the same multi-year, multi-million $ wild goose chase. As for the virus/autism stuff, it get’s worse and worse
This has been the most worthwhile thread I have read on this topic. Even Abbie didn’t use one swear word although I would agree there is too much emphasis on personalities in general. Personally, I’m not a huge Judy fan, but I think disagreement can and should be kept to the scientific.
“Lakia” asked, “…The media pay no attention to these differences between patient and healthy control viral sequences, and the different primer sets used. Did no one actually read the paper?”
Yes, journalists do read science papers – most embargoed papers are provided a few days prior so journalists can read them and contact primary sources. (Scientists who do not provide copies of their work for independent evaluation should not be taken seriously IMHO). But, are journalists virologists or biologists? Not usually just as most scientists are not usually journalists. And even if journalists are scientists there are many, many specialties within those fields – a degree in physics isn’t much help in figuring out what a lyctic cycle is.
Additionally, even if they do have a science background, journalists still have to follow professional protocols and interview experts since news articles are not editorials where the journalist’s opinion is given.
But, you are correct, it is best to use original sources for information and some background helps when determining which questions to ask. I believe Thomas Maugh II of the L.A. Times addressed primers in the telebriefing noted above although not that exact question.
Plus, this may all be a tempest in a teapot. Virologists tell us that more than one pathogen can cause the same disease. So, are other viral associations with CFS, particularly CMV, EBV and HHV-6, a dead end as some have theorized? Maybe, maybe not.
If you actually review the literature, the most accurate thing that can be said is that not all viruses, and a few bacteria/toxins too, can be found in all subsets of all patients – assuming all patients tested actually have CFS.
(As others have noted, mixing patients with non-patients using an umbrella term is a pretty good way to confound research results. Or for example, quite a few of the studies used the 1994 Fukada “cafeteria” definition requiring only duration – not severity – with only four of eight symptoms required. Theoretically, two patients could be given the same label and not have any symptoms in common and/or far different degrees of severity.)
While it would be convenient if there were but one pathogen responsible, a retrovirus “mastermind” in particular, that is not how the literature reads as of today.
Two other areas which have received very little attention are the issue of timing when it comes to sample collection and the assumption that pathogens are found only in the sera. I think mindful scientists will probably address these issues as well in upcoming months. And upcoming metagenomic studies will likely add to knowledge base as well.
Just as an aside, a press release has been issued by the Vrije Universiteit Brussel (VUB) that Belgian CFS expert Dr. Kenneth De Meirleir has positive research regarding XMRV, or would that be MuLVs, in CFS patients. However, until it is published in the peer reviewed literature it will be difficult to fully assess what has been found or not found.
True you can’t expect all journalists to be experts in the fields, PCR-ologist, virologist. Still journalist should be critical.
Although I can understand some details go to far. A journalist told me: good detailed job, but we must keep it simpeler for the reader. 😉
And I can understand her point.
I do think that different use of primers should have ringed a bell …somewhere.
With respect to the herpesviruses. I never expected them to be specific for CFS, simply because most humans are infected by them anyway. They may play a role once they are reactivated by each other, retroviruses and/or a defective immune response (or interleukins). For instance people with aids often get CMV-retinitis, due to reactivation of the latent virus.
FYI: Francis Collins, Director of the NIH (which has an annual budget upwards of $31B) will be giving the opening talk – together with Stuart LeGrice, head of the Center of Excellence in HIV/AIDS and cancer virology at the National Cancer Institutes – at the 1st International XMRV workshop this coming week. (See program here: http://www.virology-education.com/index.cfm/t/Program/vid/1FFCB7B8-FB88-C1D2-92C420E3BCEB0FB0)
This ultra-high-level attention does make me wonder what’s going on beneath the radar. In other words, I suspect Laika, that you may have more of your precise and important methodological questions answered shortly…
That podcast on Wednesday is getting more and more appealing.
@spit etc
I understand the feelings that CFS patients look for any sign of hope that there is a pathological cause of the disease but that is no excuse for the shoddy ethics involved in this for profit test (it matters not that they say all profits go back to research – what is profit? Profit could be the money left over after various individuals have taken their cut, and in any case, it is irrelevant).
The test is wrong because:
1. Paper published in Oct 2009, commercial test released in Oct 2009. Based on a few results from a few people carried out by one lab and yet sold as a 100% accurate diagnostic
2. No conflicts of interest declared in the Science paper. This is actually shocking – they obviously knew before the Science paper was published that they were going to commercialise the test.
3. Four subsequent papers did not repeat the results
4. The Lo paper did not repeat the results either as described above
5. The authors have a HYPOTHESIS about the virus and CFS, this is not the basis for a commercial test
6. There is no treatment known to work even if the virus is found
7. Anti-retrovirals will certainly be experimented with by patients and doctors. This is dangerous given the side effects. Whether they are effective or not the anti RV therapy will certainly lead to some people “feeling better”. There will be testimonials used to drive further sales. This happens with ALL CFS treatments (hair analysis, homoeopathy, etc).
8. There will be alarms about infecting family members to drive sales (and fear). In fact this already goes on, by the senior author who says in an email to an individual: “To be clear..I do think even if you tested negative now that you are likely still infected with XMRV or its closest cousin..” http://bit.ly/9OxVkS
9. And on and on
Keith,
I think you are overly cynical.
Check PubMed. There have already been a handful of positive clinical trials with anti-virals in CFS patients as well as immune modulators.
Immune modulators may be the more efficacious treatment given that anti-virals are virus specific and less effective when there is more than one reactivated virus or persistent viral infection in a patient at one time. They are also most effective in the first five years of this neuroimmune disease.
And the efficacy generally isn’t measured by subjective “gosh I feel better,” as is done with CBT trials. Clinicians generally use objective means to measure whether a drug is effective or not.
Whether you or I would agree or not, adults have the right to choose their treatment. Patients with other diseases do it all the time regardless of whether the full etiology of their disease is known or not.
Plus, think this through. The sickest patients are the one’s least likely to be able to work and thus the most likely to be unable to afford private anti-viral treatments. Insurance won’t cover it – assuming they have medical insurance left. And even in pilot trials, if they are cost recovery trials, once again only the wealthiest will be able to afford the extra $25,000+ per year in costs.
It is unlikely that there will be an overwhelming majority of patients getting such treatments any time soon – it is to be assumed that most will continue to suffer.
@KAL
My comments refer specifically to the commercial test on offer. So far, apart from the lack of confirmation by other, independent labs and the confusion illustrated in this blog post, even the WPI authors admit that they have no idea whether the presence of the virus is cause or effect (e.g. if CFS have reduced immunity in general they will be more prone to infection).
I know that in clinical trial that there is generally a very controlled analysis of symptoms and improvement and I wasn’t talking about them. I was talking about a commercial lab selling a test and physicians prescribing based on the results – if patients feel better I bet that the physician will not redo the test, given the cost and the fact that it is not covered. There will be patients who feel better and these will probably be used explicity or implicitly as cases to help sell the test. Do we want this sort of thing http://bit.ly/akYxSE ? Does it help us move forward with finding the cause and the treatments?
Here is how it should go:
a) Presence of virus needs to be confirmed by others, independently, maybe Lo has done this but things are still far from clear.
b) Epidemiological studies to look for clues of virus causality
c) Proper clinical trials of anti-retrovirals in “infected” patients, look for improvements correlating with virus disappearance
I’m not cynical, I think that the test being put on the market by the same people who published the paper and at the same time is one of the most shocking things I have seen in diagnostics.
Further – I do not either subscribe to the idea that we need to go on for 10+ years or more with clinical trials to prove clinical utility. I am very much in favour of using new knowledge when the evidence is strong enough to suggest higher benefit than risk but still a long way from the type of “proof” that insurance companies (or the NHS in the UK) would require.
Further still – if the subsequent studies had confirmed the original work, or if further studies do so, then I would in that case agree that the vipdx test would be a valid offering, even before any further epidemiology or clinical trials.
The point Laikas makes above about the autism/vaccine stuff is very pertinent here – the badly handled process cost years and $millions in progress, and also cost lives.
My bad – what my brain meant to say while my fingers were busy typing is that anti-virals, not immune modulators, are most efficacious within the first five years.
This may be because by that time many of the abnormalities that accompany this disease in subsets of patients may be irreversible at some point.
Just as an aside, many clinicians familiar with ME/CFS believe patients can be accurately diagnosed within one month not six.
Keith
Much more comfortable with the clarification. On that we happen to agree.
Until XMRV or MuLVs or XMYZ are proven pathogenic in any disease, not just CFS, then yes, tests are a waste of money outside of clinical trials. WPI definitely jumped the gun there to say the least and I am being charitable.
Longitudinal studies would be helpful, but without stratification they may be limited. Pilot studies regarding biomarkers have been done, but biomedical funding is often in short supply in this field. Fletcher et al 2010, Brenu et al 2010, Gow et al 2009, Natelson et al 2005 are just a few examples off the top of my head.
With diagnostic markers that are not specific to any other disease it would be much easier to sort patients from non-patients as well as sorting out diseases that may have core symptoms in common, but are not the same disease.
@Kal
I think that Keith makes a very good point here.
The word “Diagnosis” is crucial here. We are speaking about a diagnostic test that is sold to patients.
Then the next question is what is diagnosed. Not the illness apparently, because patients already have been diagnosed with CFS. And if it would be true that XMRV also occurs in 40% of the people with autism, and 5-10% of the normal population, then it would be rather useless for this purpose.
The only aim of the current XMRV tests is to determine whether XMRV is present……
This makes only sense, when the test is reliable, sensitive and specific (finds no or seldom XMRV in healthy controls and other diseases).
And of course it should be established if there is a causal relationship between XMRV and CFS (or most of the CFS complaints).
For a commercial diagnostic test this is certainly a requirement.
Now the results with XMRV are still controversial, not repeated and, as everybody admits, causality has not been shown.
From a patients view point, I understand that they want to try almost everything, if nothing else helps. Also, that they want to try medication that has not yet proven effective.
But a positive XMRV doesn’t solve anything, as yet. XMRV-Positive people might consider antivirals, don’t do anything, and likely just become more confused. XMRV-Negative people might still contain other retroviruses, or might have a wrong “immune signature”, etc.
What is the use? Do patients feel better?
(and I’m even not addressing the conflict of interest and the direct approach of patients)
If anti-retrovirals are to be “tested”* (and I wouldn’t object to that if XMRV or MLV are a reproducible finding), it should not be done on an individual basis (with unclear results), but in a good clinical trial –
Because it is very important to determine objectively whether there is a true effect.
ps 1. Patients shouldn’t pay for it either.
p.s. 2 @kal there is little against subjective tests in CFS patients. There are well validated questionnaires for this. A patient who feels much better is the most important outcome. And s o is a patient that would return to work. However this subjective feeling should be recorded objectively in a good trial.
I wonder which good objective tests you think would record improvement. Determining XMLRV-titers is of course objective, but meaningless. (surrogate marker if a marker at all)
Perhaps
I hope I’m not jumping the gun on an upcoming post, but @lakia you asked about biological outcome measures that can be used to determine whether say a drug is working with purportedly XMRV positive patients.
The makers of Ampligen are apparently doing just that ( yes I know, crappy company, possibly a good drug). They did a poster presentation at the recent XMRV workshop as follows: “Using exercise tolerance to test illness severity and improvement, researchers say participants who tested positive for XMRV antibodies (which suggests infection) tended to have a more significant improvement on 200-400 mg of Ampligen than those who were XMRV-negative. If this finding is substantiated in further trials, it could help doctors determine who is likely to respond well to the drug.
An interesting finding in this study is that the XMRV-negative chronic fatigue syndrome participants generally had lower baseline activity levels and abilities to complete normal daily activities. It’s too early to know whether this is consistent or accurate.”
What is good about this form of measurement is that they are measuring a symptom that is both cardinal and fairly unique to CFS. Post exertional malaise unrelieved by rest, upon very minimal exertion and lasting 24-hours or longer is a requirement not an option with the CC definition. In other words not some vague undifferentiated fatigue, but a well defined symptom.
Measurements common to every Tom, Dick and Harry of a disease and some non-diseases are much iffier as outcome measurements because they can, and in the case of CFS have been shown to, introduce research bias. The problem isn’t so much the tests, but their lack of specificity.
@keith – you addressed the problems with commercial companies promising something no one else can do – here’s a good example from PR Newswire – a nanotech company that claims to be able to diagnose tons of diseases with just one test. They refer to CFS as a stress-related disease – wonder if they are referring to viral stressors?
http://www.prnewswire.com/news-releases/early-detection-for-alzheimers-chronic-fatigue-syndrome-and-other-diseases-in-vitro-diagnostic-tests-from-neuro-biotech-corp-to-a-doctor-near-you-103277569.html
I honestly do agree with every point you make here, and I’m particularly troubled by WPI having any particularly solid connection to it whatsoever. I think that _even_ ignoring the rest of the ethical puzzle, and _even_ assuming that any worry of conflict of interest is unfounded, it opens a whole can of worms in this that should have been avoided.
I think my problem in return is that, whether it’s exactly ethical or not, there are very real problems in the way our system goes about treating both new research and difficult diagnoses/symptoms that drive a perceived need for this kind of testing — not just this, but independent labs all over the place with unregulated methods to “diagnose” patients, where their standard medical care has failed. Has it failed because they’re asking for miracles? I don’t think so, honestly. That doesn’t mean, for a second, that I want to live in a wild-west of medical care; it does mean, though, that we’re really going to have to address those other problems in order to simultaneously (and ethically!) be able to ask people to refrain from going and looking for their own, sometimes scientifically-questionable answers. Trying to provide a means for patients to do that this early was a mistake, IMO, but it’s one that I can understand — it’s muddy, for me, as a broader question.
Scientifically, though, it’s not muddy at all to me, it’s simply a bad idea. I think it is way premature to be testing patients outside of a research setting.
@spit thanks for the interesting points you raise and @keith thanks for the good explanation of the issues involved in testing.
@spit with regard to your latest comment:
whether it’s exactly ethical or not, there are very real problems in the way our system goes about treating both new research and difficult diagnoses/symptoms that drive a perceived need for this kind of testing — not just this, but independent labs all over the place with unregulated methods to “diagnose” patients, where their standard medical care has failed.
The whole point is THAT the diagnosis of CFS is difficult and no cause has yet been found. Nor has it been demonstrated that vitamin levels, hydrocortisone levels etc are affected, nor has it been shown that giving vitamins, alternative medicine etc relieves symptoms (on more than a n=1 basis, which could be a placebo effect).
As long as there is no solid basis/reason to test for it, it shouldn’t be offered. At least not -as you state correctly- out of the research setting. But everything should be done to speed up research in this area. CFS is one such disease that has never got enough attention.
It is true that regular diagnosis fails right now (and so is regular treatment), simply because no cause has been determined.
Patients, at least always ask yourself if such a test would solve anything for you. What would you do if you would test XMRV+? Or XMRV-? Would it really help you? Or would it increase uncertainty? And what about MLV’s? immunological profiles?
Lakia –
Okay, now I’m sure I really will get myself in trouble here because I am going to be very simplistic and somewhat theoretical and I write about science but I’m not a scientist:
Of course feeling better is the whole point but some measurements are more specific than others.
I hate to use any of the XMRV/MLV papers published thus far as an example since they all have problems, and I don’t wish to be mistakenly thought to be defending something I’m not, but here goes.
Lombardi et al for example used the 2003 Canadian Consensus Criteria in addition to the original 1994 Fukuda definition. This definition includes some measurable criteria.
Post-Exertional Malaise is considered a hallmark symptom of CFS where there is a pathologically slow recovery period usually 24 hours or longer upon minimal exertion unrelieved by rest.
A group at the University of Utah is looking at central fatigue in CFS. In 2009 they published a paper in the peer-reviewed literature titled, “Moderate exercise increases expression for sensory, adrenergic, and immune genes in chronic fatigue syndrome patients but not in normal subjects.” Would these measurements change for example? Of course if they did then research would have to puzzle out what that means.
You could use fatigue scales assuming they measure pathological fatigue and do not have a ceiling in this specific population. Or you could use both.
Another example is orthostatic intolerance in CFS which has been researched quite a bit by Peter Rowe at John Hopkins and is also included in the Canadian criteria. OI testing is often done using a tilt table using standardized criteria. Would these results change?
Cognitive issues in CFS have been measured in a number of ways, perhaps most intriguingly by qEEG. In unpublished work, Frank Duffy of Harvard reports that he and colleagues found that EEG data discriminated between 905 CFS patients, healthy controls and patients with depression. Would these measures change in CFS patients?
And, if they did change, at what stage in the disease process were the patients? Would that make a difference?
And if in any test there were measurable changes did they remain the same at follow up – six months, one-year, five years etc.? Don’t forget to account for LOCF problems.
The problem with questionairres is when wording is vague or wording means one thing to researchers and another to the person answering the questionnaire.
For example, if you ask CFS patients, “Do you have decreased energy?” and they have the specific symptom of exercise intolerance but not depression should they answer yes if that is the only option that even remotely comes close to what they are experiencing?
And if they say yes because post exertional malaise with a pathological recovery period could conceivably be considered “decreased energy” and the person scoring the test assumes that “decreased energy” is a symptom of depression what have you really measured?
I guess what I’m getting at is that there are many validated instruments available, but not all are appropriate or applicable and if used need to be quite precise. The less precise you are the more likely it is that a symptom or sign that can be caused by multiple variables might confound the results if you don’t rule out the others. Precision is one way of doing that.
The same with viruses, if viruses are found in both the healthy population and people with a specific disease then you have to look for the variable or variables that set one group apart from the other. EBV is one example. EBV has been closely associated with some lymphomas, but not everyone with EBV has lymphoma.
Lots of questions and variables here. It’s all quite fascinating on an intellectual level.
Keith – you said it all.
[…] NHI/FDA Paper Confirm XMRV in CFS? Well, Ditch the MR and Scratch the X… and… you’ve got MLV. Weer bij Laika (additionele artikelen zijn daar ook te vinden voor wie er nog wat verder in wil duiken). Ik volsta […]
Hi,
i like this discussion 🙂
I think the whole study situatin is a real mess at the moment. As Laika pointet out, only the WPI found XMRV in CFS Patients. But what really puzzles me is the Fischer et al study wich looked for XMRV in the respiratory tract of german patients (DOI: 10.3201/eid1606.100066). They found it in about 3% of the healthy controls and in about 10% of an immunosuppressed cohort. Well, IMO the differences between the groups arent statistically significant because the groups were way to small, but however they *found* it in every group they looked. So, why are *all* the other studies exept WPI zero/zero? Really nothing fits together here…
@formalscientist Thanks for your contribution. I like the discussion too, because most commenters are really discussing the findings and trying to reason why findings are contradictory. I can imagine that some outcomes could be 0/0 if the procedures aren’t right (material used, amplification-procedures). If the sample-size is too small (so that even with a sensitive PCR a rare positive could be missed) and if the patient population is not comparable to the population tested by WPI. But if you have a good sample, a very sensitive PCR, and a large control sample then it is hard to understand why control samples are all negative (bc controls are appr. 5% positive).
Thanks for the link. Yes the differences are not significant (well [it was] “at the 90% confidence level but not at the 95% level (p = 0.078, 1 sample t-test).) LOL I was never allowed to mention a p lower than p<0.05.
[btw the groups are not so small, but the % of infected cells are.]
i'm not sure why you find these results more odd than the discrepancies observed in patients thus far.
But anyway, it is interesting. Just striked me that they looked in respiratory samples, the tissue where the XMRV in macaques like to go. Just wonder whether it is a site where you could find XMRV in CFS-patients as well….
Laika, you say you don’t listen to highly respected virologists in the field, you look at the research itself, yet your whole post is basically a re-hash of what ERV (notoriously biased against WPI, Dr Mikovits and CFS patients) has fed you. I have to wonder if you are a “medical librarian” at the same school where ERV is a mere grad student with no scientific accomplishments that merit the amount of weight you give her opinions.
ERV has called Annette Whittemore, the CEO of WPI, a “housewife”; she calls the story of Whittemore’s daughter Andrea, who has had CFS for 20 of her 32 years a “sob story”. That is obviously what ERV thinks about ALL ME/CFS sufferers…it’s a sob story.
You accuse Alter/Lo of wrongly saying they found XMRV in CFS early in your post, but then later you admit they didn’t say that at all. Then you rehash all the former complaints against the research they have done, while giving the CDC paper a total pass. Why not at least address the issue, raised by Dr Vernon, virologist who formerly worked at the CDC, that the tubes they used to collect the blood were unsuitable for virus research. If true, it’s not surprising they didn’t find anything, and neither did NIH, in those samples. The CDC under Reeves has been using the research money they got for the research of “viral and infectious diseases” for trying to prove CFS is a mental illness, caused by childhood sexual abuse, bad mothering, and the latest from Reeves … personality disorders!
Dr Vernon said the CDC study was designed to not find XMRV.
No need for the proper chemicals in the tubes if you assume a virus is not involved before you even do the “research”. Why mention the CDC paper as if it added something to the search for the cause and treatment of CFS when in fact their cohort was found by random dialing and self described “fatigue” and didn’t have any physician-diagnosed CFS patients? Bad sample collection and a non-CFS cohort, yet you cite the CDC paper as if it were of the same quality as Alter/Lo.
Why aren’t you dissecting the study which found 30% of their bogus cohort to have personality disorders, 70% to NOT have, then came to the conclusion that CFS is caused by personality disorders! Has ERV not yet spoken to this, or is it that she likes that theory?
When you quote the Failure to Find studies as if they were of equal caliber to WPIs or the Alter/Lo/Komaroff study, you totally ignore the fact they came from psychiatrists with decades of history trying to counter the biomedical research findings on CFS. That you were apparently unaware of the studies that have found what are probably biomarkers for CFS is also telling.
Punctate lesions similar to those found in AIDS were found in brain scans of CFS patients way back in the 1980s, yet CDC called it “hysteria” then and has been promoting the psychosomatic angle ever since. Forgive me if I don’t take any research CDC does on CFS to be really looking for the biomedical cause. The time, money, opportunities and lives they have wasted is arguably criminal.
Simply counting up the studies without examining them is not illuminating. Dr McClure said she was “1000% sure” there was no XMRV in UK, yet many UK patients who tested independently tested positive for XMRV. Her 14 year-old samples were provided by Simon Wessely, psychiatrist, who promotes exercise and talk therapy as “treatments” and who has participated in having ME/CFS patients forcibly removed from their homes to mental hospitals or thrown into a swimming pool to “prove” they were faking.
You also seem to have the idea that “patients” are some group of emotional dummies who couldn’t possibly have informed opinions on the subject of their own illness. In fact, many of these patients are doctors, nurses, scientists and former biomedical researchers themselves. Many patients, even non-scientists, know more about the biology, politics and history of CFS than the average doctor, researcher or medical librarian. The inference that CFS patients are too dumb, too desperate or too emotional to withstand the “nefarious” pratices of WPI, VIPdx or the WPI researchers is just another insult to the CFS patient community. We know that neither XMRV nor the newly described MLVs absolutely prove causation. We also know that the strong association needs to be followed up and that the Alter/Lo paper would never have happened if WPI hadn’t done the work they did, and continue to do.
Other commenters inferences that the motivation of WPI/VIPdx or Mikovits is purely greed just isn’t supported by the facts. It looks like a smear campaign to me. If Grimaldi is so “shocked”, why isn’t he complaining about that testing company that put out a “test” first, using only a drop of blood on paper? VIPdx came out with their test, in part, because of that. They also said from the very beginning that it would be best to wait for more research to be done and that the test would only be useful in research, so cherry-picking comments out of context is misleading. Ignoring the original motivation of the Whittemores, to find the cause and treatment for the illness that has disbled their daughter for 20 years and for which the medical establishment has a terrible history, is misleading as well.
One very real significance of the Alter/Lo paper is that political and economic. Federal research money will now follow the leads that WPI found and viral research into CFS will not be disappeared into the political cesspool that has been federal CFS research for the last 3 decades.
@oregano I won’t waste my time on trying to invalidate arguments/ vicious remarks of someone who apparently didn’t take much trouble to read the actual post.
I just want to say one thing:
Other commenters inferences that the motivation of WPI/VIPdx or Mikovits is purely greed just isn’t supported by the facts. It looks like a smear campaign to me. If Grimaldi is so “shocked…
I’m not accusing, implying or inferring greed – but the individuals concerned have only themselves to blame. They did not disclose highly relevant potential COI’s when they should have (in the Science paper). If they have nothing to hide and yet they did hide something, the question will always be… why?
If the positive results were a result of contamination why would two completely independent studies have found 70-80% positive results in CFS patients and less than 10% in healthy controls. These were blinded studies–meaning the researchers themselves had no idea which samples came from which population and every sample got the same testing procedure. Unless I am missing something here, contamination can’t really select for patients showing symptoms of the disease vs. controls who are healthy.
Taking the 10,000 foot view here, I would expect that if there was contamination in either of these labs, it would have effected both patient samples and control samples – since they all underwent the same testing. Thus the results in the “contaminated” labs would have been much closer percentages for the patients vs. controls.
I can’t really think of an example where this wouldn’t hold true, can someone else? Its kind of the whole point of doing blinded studies (or one of them).
Also, with respect to the WPI finding “XMRV” and Alter et al finding “MLVs”, is it possible that we just don’t know enough about this class of viruses yet to know how related these are? Or perhaps, as demonstrated in the Abbot labs/Emory monkey study the “XMRV” virus is really more tissue bound and, as the WPI has said, does need culturing to bring it out. But that other MLV strains are more in the blood. Perhaps WPI looked specifically for XMRV using culture + PCR (for the Science 2009 paper) and found it, but then later (according to comments by them) broadened their search and also found MLVs? But then Alter + Lo didn’t find XMRV specifically because they didn’t culture first? Pure speculation… but curious.
This also begs the question of why these people keep doing blood studies. The Monkey model showed that the virus really didn’t spend much time in the blood after the first few weeks of infection and actually needed a provoking inflammatory event to get it back into the blood compartment after that point. They showed it settled in the sex organs, spleen, liver, GI tract, and other lymph tissue. If I were a researcher, I’d probably be looking at GI tissue in CFS patients. CFS patients are notorious for having GI complaints as part of their illness. In fact, someone did a study looking for enterovirus in CFS patients a couple years ago. Why not pull those same samples if they are still viable?
Doctors and researchers love blood tests because they are easy and cheap, but its quite possible that a reliable blood test might not be in the cards for this pathogen whereas a tissue biopsy could be much more telling.
Alter/Lo have stated that their study was not blinded.
I think that nothing was said about blinding in the original Lombardi study in science.
@rrm Exactly. Plus both use separate gels for CFS-patients and healthy controls without specificity/sensitivity controls on each gel. It may be that they normally put the patient and control samples next to each other, and this was just for the paper, but you just can’t tell.
@acer2000 and @KAL , I will come back to you later. Off to work!
I knew I read the Lombardi/Mikovitz WPI/CC/NCI study was blinded somewhere. Here it is:
http://tinyurl.com/394ttqa
This is from the May 14 2010 issue of Science where Drs. J Mikovitz and F Ruscetti wrote a letter to address some of the criticism of their paper. Starting in paragraph 5, line 8 it states:
“All samples were blinded, as mandated by the NCI and WPI institutional review board approvals. All experimental procedures were done by the same personnel, in the same physical laboratory space, under identical protocols. Investigators at NCI received 100 samples from individuals without knowing their health status; furthermore, the samples were sent to NCI directly without passing through the WPI laboratory space. Laboratory workers at the NCI and the WPI who performed the polymerase chain reaction (PCR) and immunological studies used coded, blinded samples that did not reveal the CFS status of the individuals.”
It appears that the study was blinded and the samples were sent independently to each of the 3 labs without passing through the WPI.
I’ll have to look through the notes from the conference call on the Lo/Alter paper. Do you have a link to where they stated it wasn’t blinded?
Hi @acer2000 You’re absolutely right. If samples from controls and CFS patients are treated the same from A to Z and researchers are blinded then contamination is almost excluded. As discussed they were not blinded -at least it wasn’t stated in the papers- and there are two different gels one for the CFS patients and one for the controls. So at least the researchers were not blinded there.
I think it is unlikely that all the XMRV/MLV products that ever have been found are contaminations. Although sometimes contaminations end up in your primer/reaction mix and then (almost) everything turns positive. (after PCR amplification products are everywhere, also in the air, in your hair etc. You need to work in a hood and in separate rooms to prevent that; in addition some people are very careless with positive controls).
Besides PCR-contamination others also fear contamination of cells with retroviruses.
XMRV being more cell-bound than MLV viruses is a good suggestion, but it is based on the idea that WPI needs to culture the virus first, which was not mentioned in their first Science paper.
Yeah I know it is just speculation, but that is what we all do if we don’t now the truth.
Your remarks about blood-testing are to the point. Commented on it elsewhere.
[…] can also be target of hate mail or aggressive comments. In the whole XMRV-CFS torrent, patients seem to almost “adore” positive journalists (i.e. Amy Dockser Marcus of the […]
Sorry no hate mail, but CFS patients have also voiced anger about Marcus at times.
Hate mail is always bad.
In general -when I read Amy’s posts- they are followed by admiring and positive comments by patients.
Most of the comments on critical pieces are followed by very negative, sometimes aggressive reactions.
It is not an absolute 100%/100% thing.
@acer2000
From the FAQ to the Lo/Alter study at the CFIDS site:
“Q: Were the samples tested under blinded conditions?
A: We did not specifically blind and mix the two sample groups (CFS and blood donors). However, they were studied in parallel by polymerase chain reaction (PCR). As shown in the figures of the paper, those samples with positive amplicons of the predicted sizes were all sequenced; no sample was considered positive unless sequencing confirmed PCR reactivity. – H.J. Alter, MD, MACP and S‐C Lo, MD, PhD”
@acer2000 (and @rrm).
“I knew I read the Lombardi/Mikovitz WPI/CC/NCI study was blinded somewhere. Here it is:
http://tinyurl.com/394ttqa
Good, that you looked this up, @acer. And you are right but what worries me that it is in a letter in Science a half year after the initial paper has been published
The problem is that a lot is published or said AFTER the publication of the Science paper. All details should be IN that Science paper. That is what peer reviewed papers are for: so that you can read all details about how the search has been done. And that you can try to reproduce the findings on basis of what has been published.
What you see thereafter at meetings, in emails, to patients or in other papers doesn’t really matter. It should be in the paper or it should appear as erratum. Meetings are good to explain the findings or to discuss them.
It doesn’t say in the October Science paper that researchers were blinded
Furthermore when you look at the one PCR-figure published, it shows one gel with CFS patients and one with healthy controls.
Pretty good random distribution isn’t it?
(ok it might have been done just to show a nice clear picture, after unblinding the samples, but then it should be mentioned that it was done only for this purpose, shouldn’t it?)
If you don’t mention it, like Lo, we just assume it hasn’t been done. That’s ok (although it should have been done, but he admits it – at least).
Lombardi et al have omitted/changed more details in their paper. And -I repeat myself- this makes me suspicious. Can I rely on what they say if they continuously change what they are saying?
1. Blinding PCR (not in paper)
2. Need for culturing (not in paper, at least not for doing PCR)
3. PCR is weak (not in paper, where strong bands show on the gel after one round (normally weak bands after two rounds of logarithmic amplification)
4. immunological abnormalities were part of the CFS diagnosis (in paper, later rectified)
5. “Samples were selected from several regions of the United States where outbreaks of CFS had been documented (S2)” is in the Lomardi paper, later it was said that these CFS patients comprised 25% of the researched population ,which is still quite a lot.
6. Only XMRV detected (unpublished: also other MLV’s)
7 Very homogeneous XMRV-sequence (unpublished: heterogeneous)
2 and 3, 4 and 5, 6 and 7 may be related.
Furthermore in their comment they state it isn’t a case-control study. But it is : you have cases, you have controls and you’re looking for an association with a virus.
It is very important that this population is representative for CFS patients or -if it is not- that it the characteristics & geographic distribution of the patients is clear. It could explain differences between incompatible results (but other things like technique are also important).
The next thing to investigate is whether XMRV/MLV presence is specific for CFS (Judy states that it also occurs in autistic people for instance) and whether there is a difference between CFS-patients (qualitatively or quantitatively)
Assuming the findings are true ( I expect some fire with so much smoke).
Yeah it bothers me too that the original paper wasn’t as organized as it could be. I mean they could have lined up their results in a table and it would have been much easier to read. Its incredibly hard to figure out which experiment was done on what sample the way they have it set up. The document could use more structure. As it is now it reads more like a narrative. They should have been more specific about their methodology. However, it was essentially a pilot study. An extensive one at that.
I guess what I am saying is, it doesn’t really matter that the info about blinding, clarification about patient selection, etc.. came out later. What matters is what *is*. If the study was blinded, it was blinded–no matter how annoying the omission of that detail was from the original manuscript. I guess maybe I am falling back on more an appeal to the prestige of the journal and peer review process, but I assumed that Science would not accept a paper that wasn’t blinded. Maybe thats a bad assumption to make, as PNAS apparently didn’t require that of the Lo paper, and it would have been easy to do.
Something that came up several times in the recent XMRV workshop in the U.S. was:
“Very interesting in this regard were comments by the head of the blood working group at the NIH who is trying to determine the cause of the discrepancy.
He hinted strongly that it is the way blood is collected and processed for nucleic acids and not the detection methods for XMRV itself that divides the two groups.
In an NIH blood group sponsored study, a group comparison study with both camps represented detect successfully, in a blinded fashion, XMRV spiked buffer in varying concentrations but they nevertheless divide into two camps when clinical blood samples are taken and processed for XMRV nucleic acids.”
However, without knowing more it was difficult for me to assess “Failure to detect xenotropic murine leukaemia virus-related virus in Chinese
patients with chronic fatigue syndrome” – published September 13 – in regards to this information.
Perhaps you are better able to judge.
Hi @kal. I’ve seen it and plan to write a new post about it. 😉 Unfortunately I’m rather busy with my regular job right now, so it has to wait.
It is extremely interesting, though not unexpected.
Of course without the actual data, it is as hard for me as it is for you to judge the results.
You know, it isn’t surprising at all that XMRV in buffer is easy to PCR. There are just 10 or 100 or maybe 1000 copies in buffer. No competition with other sequences, and tadadada you can easily amplify XMRV in the test tube. That is quite different when you have 10 or 100 copies of XMRV in BLOOD and especially if you didn’t purify white blood cells (PBMC) first (there often PCR-inhibitors in bodily fluids). The DNA in the PBMC compete for the primers and they might win if they are abundant and the virus is not and/or if the primers are not so specific etc.
It is just not fair to use XMRV in buffer as a positive control. You have to make a dilution in PBMC if you test XMRV in PBMC, you have to make a dilution in blood if you test XMRV in whole blood. Otherwise you can never say something about the true sensitivity of the test (that is the sensitivity in the same conditions as you use for testing the CFS samples).
Therefore I’m not surprised that if you amplify XMRV from the normal patient samples you can get a negative result even if your PCR from XMRV in the buffer was positive.
There were some negative studies that did very good spiking experiments though. Lombardi did not.
I hope that I made myself clear enough. Please tell if I didn’t. It is a tough subject.
Surprisingly I did follow you. 😀 I just watched a press conference with retrovirologist Myra McClure talking about why she believes there is not a retroviral link with CFS patients at this time.
Bio-politics aside, she had interesting points – some of which has also been made by ERV.
Of course no one can know everything. She was asked about all of the studies published thus far focusing on blood tissues instead of say cerebral spinal fluid. Obviously CSF is much more invasive than drawing blood and she said there wasn’t a reason yet to do that. But there might be. Retrovirologist Ila Singh is doing XMRV work in autopsy tissue samples from CFS patients that will be no doubt interesting either way.
The same clinician who commented about the blood processing comments from the workshop in Bethesda also noted a presentation regarding animal studies. He stated, ” a very interesting presentation was made by a group connected to Abbott Labs that infected male and female Macaques (monkeys closely related to man) with human XMRV to see what happens and where the virus ends up or concentrates itself.
Within a few weeks, the virus was largely cleared from blood where it was initially injected in high concentration. Even antibody response was lost over time (months) as the infection was largely removed from the blood and virus did not appear to persist in the blood.
Apparently, there was not enough viral antigen to keep antibody levels high or persistent. However, the virus was found more or less in every organ, at least initially, and thought to be carried around the body in T-cells and B-cells during the active phase of infection.
This is consistent with the ubiquitous nature of the Xpr1 receptor used by the virus to gain access to almost all cells of the body.
Organs where the virus was initially most concentrated appeared to be lymphoid organs such as the spleen, liver and mesenteric nodes of the GI track and in sex organs and in particular the epithelium of the prostate gland where it was highly concentrated at first and then the infected cells later apoptosed and infection disappeared from the epithelium and then the virus was more likely to be seen in the interstitial cells in the stroma or matrix of the prostate, especially the fibroblasts which may be one reservoir in all the various organs that are initially infected. The virus was also found in the epithelium of the cervix in the female macaque.
(Say that ten times fast!)
Over time, the infections of various organs tended to be cleared by either immune mechanisms but especially by restriction enzyme systems present in almost all human cells that hypermutate the virus so it cannot persist as a competent infectious agent.
Indeed, mutated viral strains are almost always found in CFS cases by both Judy Mikovits at WPI and Frank Ruscetti at NCI. Sometimes this makes the virus incompetent as an infectious agent and sometimes has no effect on infectiousness.
A Tissue Reservoir – Very interesting is that another cell that appears to be a reservoir of XMRV other then fibroblasts within tissue stroma are tissue macrophages
The pulmonary alveolar macrophages were absolutely loaded with XMRV virus and other tissue macrophages could also be a potential reservoir in other tissues as well, especially in the GI tract, sex organs and sinuses.
Tissue macrophage reservoirs would be analogous with HIV as well. It would seem that bronchial secretions, nasal secretions and sex organ secretions as well as feces and urine are well positioned to help the virus to spread itself to other macaques, especially if activated.
As for activation of more or less low level or quiescent but persistent infectious virus, there seem to be several mechanisms.
The virus has both a glucocorticoid response element (GRE) and an androgen response element (ARE) in its promotor region. It also has binding regions for NK Kappa B proteins in its response elements. In any organ with high levels of local androgenic stimulation such as the prostate and perhaps during puberty, the virus could activate.
Activating XMRV: Stress and the Hormones – No mention was made of the effect of the predominant female sex hormones but estrogen is the equivalent androgen-like hormone in females.
As for the GRE in the promotor region, severe stress will activate the virus or the use of glucocortocoid hormones and perhaps any precursor steroid hormone such as pregnenolone.
As for the NF Kappa B sites, any strong immune response with an associated cytokine storm would also be a strong stimulant and such stimulation certainly occurs in the bronchial tree which is frequently stimulated with antigen, especially during allergy season.
Perhaps most interesting of all was what happened with the injection of a bolus of foreign peptides into macaques that had apparently completely cleared the virus from blood.
There was a huge reactivation of infectious virus in the blood proving that latent but persistent virus is just below the surface and that XMRV infection cannot be completely cleared from all reservoir sites. The peptide injection mimics an acute infection (? borrelia or the flu), an immunization or even acute mold exposure.
The effect of XMRV infection over time was not studied in the macaque but a similar gammaretrovirus called Feline Leukemia virus (FeLV) has been well studied in cats for decades.”
Fascinating stuff.
Haha. Lol. Glad you did. Hope others do too.
Ahum, yes I read it. Are you giving away my next posts? 😉
Regarding Mc Clure, I just must say she had the worst PCR-results of all. But that doesn’t mean she doesn’t say sensible things. I did see the video mentioned on twitter, but do you have the link perhaps? It is good to hear what she has to say.
With respect CSF: you’re not going to do such an invasive test, unless you’re pretty sure that is the site the virus is (and nowhere else).
Till now if the the virus was found, it was found in the blood (and in the urine in case of prostate cancer). So it is only logic that people first try to reproduce that. Also because it is easy to get (as you say) and there is a lot of (frozen) archival material.
But since most patients have a failing immune response, many different complaints and considering the macaque-experiments it would be very interesting to look at the distribution of the virus in people with CFS (and others), i..e in people who died. Preferentially not in archival material, because that may be harder to PCR (if that is the technique of choice).
It would explain why Mikovits had to culture the PBMC (had she only written this from the start, it would have been much more plausible).
On the other hand Lo et al found the most prominant MLV- (or PMRV-) signal in the plasma.
Ooh and the macrophage, which microbe doesn’t it harbor? (btw PBMC contain monocytes which can become macrophages upon leaving the bloodstream.)
It’s terribly complicated.
But I think it is a very good thing that the NIH is doing a trial, to find out where the shoe pinches.
It is also good that CFS patients do get more recognition. I hope some one or more culprits of this disease can be found..
p.s. I see @acer also quotes the macaque experiments and addresses that the virus might be present in larger quantities at other sites than in the blood
Here’s another McClure interview about XMRV (recorded yesterday I believe):
http://www.medpagetoday.com/MeetingCoverage/ICAAC/22166
@acer2000
I think it is not a very good assumption to make regarding the Science study. The paper is reviewed based on the manuscipt itself. There is no way reviewers demanded proof of blinded testing without this being mentioned in the paper. If anything, it confirms that Science was more interested in the XMRV part than in the CFS connection (I believe it was reported by Mikovits that Science actually requested a title change, one without the term “CFS”).
@RRM and @acer2000
I have to agree with Mark (@RRM). Sorry but this is a full research paper, peer reviewed, in SCIENCE (WTF)! If there are uncertainties regarding the outcome – on such a controversial and important issue – all care should be taken to present rigorous data and rigorous methods, described in EVERY detail.
How do we know if it is true what they say afterwards? Why do the gels show patients on one gel and the controls on the other- if all results are blinded? The samples also were from different places/occasions. If it is true that handling of samples is the most important variable in finding XMRV this is also crucial to know.
This detail isn’t the only detail the “forgot” to mention (see previous comment).
So in peer reviewed papers it *does* count what is said IN the paper. Per definition! Otherwise we rather not write it down but just write blogs and make videocasts.
If things are not properly mentioned, the reviewers are partially to blame. They should demand all details. I would have asked for sensitivity controls and spiking experiments and blinding. (i wouldn’t have asked if you needed to culture the cells before PCR-ing because there was no reason to believe it was needed. -strong bands after one round, direct isolation from PBMC).
I do not think the reviewers did a great job. Perhaps it is as Mark said: Science was more interested in the XMRV part than in the CFS connection. I also think the reviewers were not very experienced in PCR, because otherwise they would have asked other controls (mere water as negative control isn’t good enough, spiking experiments, blinding)
Great additions to the discussion since I’ve been missing.
I read about and found the macaque thing very interesting, too; it makes tons of sense, of course, to look in blood since that’s where we’ve found stuff (and _much clearer_ published methodology in the original paper would have saved time and many bits of internet arguing in this regard, frankly), but if the levels in blood change over time or over the course of the disease, then we’re going to be in for a heap of mess right now unless we look at other tissue samples that are potentially more consistent. That’s a distinct possibility here, I think, though there are many other potential factors to consider, of course. I’m still not convinced that we’ve even begun to tease out the possibility of diversity in target loci or consistent methods across the samples already studied; there are a lot of questions still in just knowing we’re finding it if it _is_ there, regardless of associations with anything. I look forward to much clarification on that, the sooner the better. If this thing (or family of things) is at all polytropic, or (like FeLV) spreads at all in feces and saliva, and causes anything nasty in humans at all, we’re in for a heap of hurt trying to slow it down. I mean, the potential implications are jaw-dropping.
Re: looking at CSF, I’d bet you anything there’s a small supply of stored CSF samples from diagnosed CFS patients who were screened through spinal tap for low/undetectable hypocretin/orexin (which would be considered positive for narcolepsy with cataplexy, which I just _happen_ to know stuff about :). It’s a troublesome thing, though — given the clinical presentations, I strongly suspect that as more physiological understanding of both diseases comes, we’ll also find that there’s been some uncomfortable overlap in the diagnoses, so there’s also a corresponding potential for some serious confounding. MOST narcoleptics with cataplexy screen fine this way, but SOME don’t (5 – 30% with clinical cataplexy, depending on the research you read), and where their diagnoses go from a negative CSF screen depends totally on their doctors; many wind up with various psych diagnoses for a time, some wind up with CFS, some are rediagnosed later. It’s another part of the mess generally, really — CFS is probably currently encompassing people with several different flat-out physiological conditions, without even getting into the possible somatoform ones.
(Testing the CSF of CFS patients = more potential for confusing typos than ever before known in medical research)
Spinal tap is invasive, and I agree that we’re not there yet, but I’d bet you they’d have no trouble finding patients willing to participate if it looks likely to answer some otherwise sticky questions. And I’ll be very surprised, given the symptoms we see in severe cases, if there isn’t functionally major but potentially minutely-targeted neurological involvement in CFS. But I’d really like to see methodology refined generally, I guess, before moving too far from there — I don’t expect every paper to be perfect, but there’s going to be no end to the confusion until we know what we’re looking for and feel confident that we can find it in real-life tissues of humans who carry it, regardless of any particular disease process it may or may not cause. That’s important — hugely important, for the patients it may effect, who I fully support in pushing and wrangling and advocating for themselves — but we also have to feel confident in the data itself before we’re going to know what the hell we’re doing. I have no such confidence, currently, and that’s step one in everything that follows.
@spit – There actually may be some CSF from CFS patients available if something like that can be reliably stored. Ben Natelson did quite a bit with CSF in CFS patients back in ’05 and ’06 I think. This was back before Fauci pulled funding for CFS Centers of Excellence that both Nancy Klimas and Ben Natelson ran. Others were also looking at proteomics as a means of finding a biomarker in CFS and related diseases.
Of course, right now we don’t even know if XMRV or MuLVs are pathogenic and they may only be found in subsets of patients – which doesn’t mean they are not causative if only found in subsets, it might just make them only one of many pathogens that can trigger the same disease and or similar diseases with core symptoms in common.
Much to tease out.
Dr. McClure’s remarks from the ICAAC press conference can be found here. The webcast video sticks a bit at first but once it gets to Dr. McClure it improves:
http://www.microbeworld.org/index.php?option=com_jlibrary&view=article&id=4778&Itemid=54
The WPI were talking about co-cultuing and which methods were the most sensitive back in October 2009. Take a look at this video of the Chronic Fatigue Advisory Committee meeting from the 29th October 2009. There is also a slide 4min in.
Lo et al. also used the same inner primers as Lombardi et al., as you can see from this quote.
“By nested PCR assays targeting the MLV-related virus gag gene, using both the previously described primer sets (3, 4) and an in-house–designed primer set with highly conserved sequen- ces from different MLV-like viruses and XMRVs”
One issue which may be confounding the results is the sample preparation used. This was also mentioned as something the blood advisory committee had just discovered, at the XMRV International Conference.
The original paper does mention culturing with LNCaP cells. Underneath figures 3 and 4.
I must not understand your point laikaspoetnik, because coculturing is mentioned throughout the paper, and in the supporting material that was also published on the 8th October 2009.
You make the following points laikaspoetnik
4. immunological abnormalities were part of the CFS diagnosis (in paper, later rectified)
5. “Samples were selected from several regions of the United States where outbreaks of CFS had been documented (S2)” is in the Lomardi paper, later it was said that these CFS patients comprised 25% of the researched population ,which is still quite a lot.
Firstly, immunological abnormalities were never mentioned as part of the diagnosis for CFS patients in the original paper. They only say that such patients can display such abnormalities. Secondly, at no point in the paper do they state that patients came only from an outbreak.
GAM,
I will try to address most of your comments in one post.
1. (Co-) culturing
As explained anything not mentioned in the Science paper doesn’t count.
Co-culturing is mentioned in the paper but NOT in the context of PCR. Thus for instance co-culturing of PBMC with the prostate cancer cell line LNCaP was done to show that the CFS-derived PBMC could INFECT the LNCaP. Results of these infectivity experiments are shown in Figs 3 and 4. These are NOT PCR experiments.
The paper as well as in the supporting material (describing the Methods more in detail) do not describe culture as a prerequisite nor a step whatsoever in the PCR.
Article: “we isolated nucleic acids from PBMCs and assayed the samples for XMRV gag sequences by nested polymerase chain reaction (PCR) (5, 6).
Fig 1: XMRV sequences in PBMC DNA from CFS patients. Single-round PCR results for gag, env, and gapdh sequences in PBMCs of (A) CFS patients and (B) healthy controls
Supporting material: The PBMC (approximately 2 x 107 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 ºC for further culture and analysis or resuspended in TRIzol (Invitrogen, Carlsbad, CA) and stored at -80 ºC for DNA and RNA extraction and analysis. ( meaning that PBMC were directly treated with TRIZOL and not cultured before treating them with TRIZOL – a chemical solution used in RNA/DNA-extraction –)
2. Primers Lo et al
Sorry but that is exactly what I said/meant. So Lo et al always used the same outer primers as Lombardi et al, and sometimes the same inner primers. In other cases (not clear when) more conservative primers were used, that Lombardi et al didn’t use. The use of these conservative primers might explain why Lombardi et al were originally not able to detect MLV (PMRV). It doesn’t explain why Lo et al didn’t find XMRV (use of both the Lombardi primers and conservative primers who would fit both)
I presume there is a reason Lo et al made these conservative primers. They could infer from the known MLV sequences, that their primers would fit not only to XMRV but also to other MLV’s.
3. the origin of the CFS samples
You object to two of my points ( 4 and 5) mentioned in a comment:
by saying:
My source for saying this was the answer of Judy herself (!) to criticism of several scientists to the Science Paper. This is Judy’s comment:
In the supporting material to their first paper, Lombardi et al said:
So in other words: Lombardi et al at least were not very careful in their wording in the Science paper. 25% of the outbreak regions (later corrected figure) is still a lot! (and not representative of all CFS patients)
Other scientists frequently refer to material presented in Lisbon (7. J. A. Mikovits, presentation at Conference on Cellular and Cytokine Interactions in Health and Disease, Lisbon, Portugal, 17 to 21 October 2009).
Here they apparently have said some contradictory things. Has anyone access to what was said there? Any CFS patients who have transcripts?
I get your point about the coculturing.
As for the patients, they may have come from areas where there were outbreaks, but they do not say they became sick during an outbreak. CFS is known to occur in both sporadic and epidemics, and there is little data on the split each type accounts for. Therefore it is not known what would be representative of all CFS patients.
Mikovits and Ruscetti may say that they regret that they implied they diagnosed patients by certain abnormalities. It is clearly not what they meant, and would still have no effect on diagnosis of CFS by Fukuda or the Canadian criteria.
Only a couple of scientists have claimed that conflicting information was presented at the Lisbon conference. Myra McClure and Simon Wessely are two, and they included this in an editorial to accompany the BMJ study. However, I’m not sure if they were at the conference. This appears to more a case of chinese whispers.
@GAM
😉
You don’t think that outbreaks (if there were any) aren’t relevant bc it isn’t know what is representative for the disease.
I do think that the chance of finding a virus like XMRV is greater in people who became sick after an outbreak. At this moment we don’t know what causes the discrepancies. Different exposure to different pathogens might be just one such difference between patients determining whether you find XMRV or not.
How can they make so many mistakes in one article: that ‘s my main point. “It is clearly not what they meant”. Ooh? You really have to check all details before you submit a scientific paper. Again they are very sloppy. Not a good sign.
Yes, that’s why I really wanted to see the data myself.
Sorry, I always do this. Never finish what I am saying in one post.
Lo et al. may not have found XMRV, because they did not culture the samples.
I realise why you say the video does not count, but I think it is important to at least recognise that they were saying the same things back in October 2009. Months before any negative papers.
The remarks from the youtube video were made about a subsequent test by the NCI, involving new patient samples. They still didn’t have a problem finding XMRV, only using PCR, in a whopping 60% of CFS samples (or in an even more imprerssive 69% of samples confirmed positive for XMRV by culture), which is in line with Frank Ruscetti’s presentation at the recent XMRV workshop.
How is it possible that one group can routinely detect XMRV in at least a majority of samples with just using PCR, when no other lab in the world can validate these results using the same method?
The Science group could be using the most sensitive PCR test, but that is highly doubtful. In the first fase of testing of spiked blood samples in the blood working group, WPI/Mikovits appeared to have the least sensitive (and the least specific) PCR assay. Furthermore, while CDC/Switzer and FDA/Lo used the very same assays that they had used in their respective studies, WPI/Mikovits decided not to test these (spiked) samples with their “Science” PCR assay but with another assay. This doesn’t make sense when your working hypothesis is that your original assay is the best in the world in detecting XMRV in the blood of CFS patients.
Whatever the solution may be, in my opinion it isn’t the cohort and it isn’t the culturing.
Although not one single major explanation for this “zone of chaos” seems very likely at this moment, that major explanation has to be out there. I don’t have any credentials in this field of science, but my intuition says it may be the tubes used, as crazy as this sounds. A bit like Suzanne Vernon said after the CDC study, but then the other way around:
All (?) positive studies seemed to have used heparin tubes. One negative study (Brigitte Huber) used heparin tubes and found XMRV because of contamination. Suzanne Vernon criticized the CDC/Switzer for not using these heparin tubes, but it may very well be that these tubes are the reason for false positives. A different method of collecting/storing samples of healthy controls, medication/nutrition differences between CFS patients and healthy controls, or confirmation bias may subsequently be the reason these lab artifacts are more likely to be reported in CFS patients than in healthy controls.
Very good points @RRM.
I never got the impression that the PCR of Lombardi et al was particularly strong. They didn’t have the right sensitivity/specificity controls either. Some groups with negative results had far more sensitive PCR assays.
That the WPI use other conditions and had the least sensitive PCR (in the spiking test) is remarkable.
My guess is that Lombardi et al had (PCR) contamination in their very first PCR’s (or Cleveland Clinic had) This would explain the very strong HOMOGENEOUS PCR bands after just one round and their failure (?) to reproduce this.
Later they might have needed culturing the cells before doing PCR -especially because their PCR wasn’t optimal.
Assuming that XMRV and MLV in humans are *real*, a number of conditions could determine whether results are strongly positive, weakly positive or negative. I think that blood preparation is just one of them.
I don’t think it is simply the culturing either. The cohort might also play a role, together with sample preparation, primers and further conditions.
I agree that the different method of collecting/storing samples of healthy controls and CFS patients might be another important variable.
(at least explaining why CFS are XMRV+ and controls XMRV-).
Could you explain to me, why heparin-tubes would contain contaminating material. Mouse DNA? You can’t assume world-wide PCR contamination, can you?
So in conclusion I don’t think that there is ONE single reason why results differ.
@GAM
😉 Never Mind. Better then loosing your complete comment after completing it (happens often to me)
Everything is possible, but I don’t think it is the most likely possibility because the WPI PCR was strong in the beginning without culturing cells. (you know you agreed that (co-)culturing was NOT a prerequisite for a positive PCR in the Science paper).
Ok now I get your point. October 2009 there was no need to defend their results, because there were no negative papers yet. But suppose they were suddenly not able to reproduce the strong PCR results they showed in the Science paper. Couldn’t that be the trigger?
[…] Does the NHI/FDA Paper Confirm XMRV in CFS? Well, Ditch the MR and Scratch the X… and… y… (laikaspoetnik.wordpress.com) […]
Sorry – what I intended to say was that WPI/Mikovits had the least sensitive test of those that had published studies thus far (Lo/Switzer/Mikovits.
Anyway, in the second fase of studies for the blood working group, WPI have collected blood from 4 XMRV+ patients from the Science paper (confirmed positive by PCR, serology and viral isolation). These samples have subsequently been distributed to (most of) the other labs for blinded testing.
The results of this second fase were scheduled to be released in August/September, but they have been delayed. John Coffin of the blood working group has said in the most recent issue of Science the reason is that “we’re still confused by [the results]”.
Makes you wonder what those results are….
An excellent post. So speaking as a non-virologist, am I right in thinking that the key question now is – why did the WPI find XMRV with their, XMRV-specific primers, when according to Lo & Alter, there is no such DNA present in either patient or control samples (although there is lots of related MLV DNA)?..
[…] PNAS, has no direct plans to retract the paper of Alter et al reporting XMRV-like viruses in CFS [discussed in 18]. Schekman considers it “an unusual situation to retract a paper even if the original […]
[…] been plenty of excellent coverage of this new study, but most of it has come from specialists and has assumed a certain degree of […]
[…] Jacqueline’s Blog – Jacqueline is a medical librarian with some experience in this field. She’s written a series of rather technical blogs on XMRV. Warning – she’s not been particularly happy with the WPI, in fact, she’s not particularly happy with anyone (although she did like the Alter paper); what she offers is a deeper dig at the technical aspects of the studies than most bloggers offer. […]