Science Asks to Retract the XMRV-CFS Paper, it Should Never Have Accepted in the First Place.

2 06 2011

Wow! Breaking!

As reported in WSJ earlier this week [1], editors of the journal Science asked Mikovits and her co-authors to voluntary retract their 2009 Science paper [2].

In this paper Mikovits and colleagues of the Whittemore Peterson Institute (WPI) and the Cleveland Clinic, reported the presence of xenotropic murine leukemia virus–related virus (XMRV) in peripheral blood mononuclear cells (PBMC) of patients with chronic fatigue syndrome (CFS). They used the very contamination-prone nested PCR to detect XMRV. This 2 round PCR enables detection of a rare target sequence by producing an unimaginable huge number of copies of that sequence.
XMRV was first demonstrated in cell lines and tissue samples of prostate cancer patients.

All the original authors, except for one [3], refused to retract the paper [4]. This prompted Science editor-in-chief Bruce Alberts to  issue an Expression of Concern [5], which was published two days earlier than planned because of the early release of the news in WSJ, mentioned above [1]. (see Retraction Watch [6]).

The expression of concern also follows the publication of two papers in the same journal.

In the first Science paper [7] Knox et al. found no Murine-Like Gammaretroviruses in any of the 61 CFS Patients previously identified as XMRV-positive, using the same PCR and culturing techniques as used by Lombardi et al. This paper made ERV (who consistently critiqued the Lombardi paper from the startlaugh-out-loud [8], because Knox also showed that human sera neutralize the virus in the blood,indicating it can hardly infect human cells in vivo. Knox also showed the WPIs sequences to be similar to the XMRV plasmid VP62, known to often contaminate laboratory agents.*

Contamination as the most likely reason for the positive WPI-results is also the message of the second Science paper. Here, Paprotka et al. [9]  show that XMRV was not present in the original prostate tumor that gave rise to the XMRV-positive 22Rv1 cell line, but originated -as a laboratory artifact- by recombination of two viruses during passaging the cell line in nude mice. For a further explanation see the Virology Blog [10].

Now Science editors have expressed their concern, the tweets, blogposts and health news articles are preponderantly negative about the XMRV findings in CFS/ME, where they earlier were positive or neutral. Tweets like “Mouse virus #XMRV doesn’t cause chronic fatigue #CFS http://t.co/Bekz9RG (Reuters) or “Origins of XMRV deciphered, undermining claims for a role in human disease: Delineation of the origin of… http://bit.ly/klDFuu #cancer” (National Cancer Institute) are unprecedented.

Thus the appeal by Science to retract the paper is justified?

Well yes and no.

The timing is rather odd:

  • Why does Science only express concern after publication of these two latest Science papers? There are almost a dozen other studies that failed to reproduce the WPI-findings. Moreover, 4 earlier papers in Retrovirology already indicated that disease-associated XMRV sequences are consistent with laboratory contamination. (see an overview of all published articles at A Photon in the Darkness [11])
  • There are still (neutral) scientist who believe that genuine human infections with XMRV still exist at a relatively low prevalence. (van der Kijl et al: xmrv is not a mousy virus [12])
  • And why doesn’t Science await the results from the official confirmation studies meant to finally settle whether XMRV exist in our blood supply and/or CFS (by the Blood Working Group and the NIH sponsored study by Lipkin et al.)
  • Why (and this is the most important question) did Science ever decide to publish the piece in the first place, as the study had several flaws.
I do believe that new research that turns existing paradigms upside down deserves a chance. Also a chance to get disproved. Yes such papers might be published in prominent scientific journals like Science, provided they are technically and methodologically sound at the very least. The Lombardi paper wasn’t.

Here I repeat my concerns expressed in earlier posts [13 and 14]. (please read these posts first, if you are unfamiliar with PCR).

Shortcomings in PCR-technique and study design**:

  • No positive control and no demonstration of the sensitivity of the PCR-assay. Usually a known concentration or a serial dilution of a (weakly) positive sample is taken as control. This allows to determine sensitivity of the assay.
  • Aspecific bands in negative samples (indicating suboptimal conditions)
  • Just one vial without added DNA per experiment as a negative control. (Negative controls are needed to exclude contamination).
  • CFS-Positive and negative samples are on separate gels (this increases bias, because conditions and chance of contamination are not the same for all samples, it also raises the question whether the samples were processed differently)
  • Furthermore only results obtained at the Cleveland Clinic are shown. (were similar results not obtained at the WPI? see below)
Contamination not excluded as a possible explanation
  • No variation in the XMRV-sequences detected (expected if the findings are real)
  • Although the PCR is near the detection limit, only single round products are shown. These are much stronger then expected even after two rounds. This is very confusing, because WPI later exclaimed that preculturing PBMC plus nested PCR (2 rounds) were absolutely required to get a positive result. But the Legend of Fig. 1 in the original Science paper clearly says PCR after one round. Strong (homogenous) bands after one round of PCR are highly suggestive of contamination.
  • No effort to exclude contamination of samples with mouse DNA (see below)
  • No determination of the viral DNA integration sites.

Mikovits also stressed that she never used the XMRV-positive cell lines in 2009. But what about the Cleveland Clinic, nota bene the institute that co-discovered XMRV and that had produced the strongly positive PCR-products (…after a single PCR-round…)?

On the other hand, the authors had other proof of the presence of retrovirus: detection of (low levels of) antibodies to XMRV in patient sera, and transmissibility of XMRV. On request they later applied the mouse mitochondrial assay to successfully exclude the presence of mouse DNA in their samples. (but this doesn’t exclude all forms of contamination, and certainly not at Cleveland Clinic)

These shortcomings alone should have been sufficient for the reviewers, had they seen it and /or deemed it of sufficient importance, to halt publication and to ask for additional studies**.

I was once in a similar situation. I found a rare cancer-specific chromosomal translocation in normal cells, but I couldn’t exclude PCR- contamination. The reviewers asked me to exclude contamination by sequencing the breakpoints, which only succeeded after two years of extra work. In retrospect I’m thankful to the reviewers for preventing me from publishing a possible faulty paper which could have ruined my career (yeah, because contamination is a real problem in PCR). And my paper improved tremendously by the additional experiments.

Yes it is peer review that failed here, Science. You should have asked for extra confirmatory tests and a better design in the first place. That would have spared a lot of anguish, and if the findings had been reproducible, more convincing and better data.

There were a couple of incidents after the study was published, that made me further doubt the robustness of WPI’s scientific data and even (after a while) I began to doubt whether WPI, and Judy Mikovits in particular, is adhering to good scientific (and ethical) practices.

  • WPI suddenly disclosed (Feb 18 2010) that culturing PBMC’s is necessary to obtain a positive PCR signal.  As a matter of fact they maintain this in their recent protest letter to Science. They refer to the original Science paper, but this paper doesn’t mention the need for culturing at all!! 
  • WPI suggests their researchers had detected XMRV in patient samples from both Dr. Kerr’s and Dr. van Kuppeveld’s ‘XMRV-negative’ CFS-cohorts. Thus in patient samples obtained without a culture-enrichment step…..  There can only be one truth:  main criticism on negative studies was that improper CFS-criteria were used. Thus either this CFS-population is wrongly defined and DOESN’t contain XMRV (with any method), OR it fulfills the criteria of CFS and the XMRV can be detected applying the proper technique. It is so confusing!..
  • Although Mikovits first reported that they found no to little virus variation, they later exclaimed to find a lot of variation.
  • WPI employees behave unprofessional towards colleague-scientists who failed to reproduce their findings.
Other questionable practices 
  • Mikovits also claims that people with autism harbor XMRV. One wonders which disease ISN’t associated with XMRV….
  • Despite the uncertainties about XMRV in CFS-patients, let alone the total LACK of demonstration of a CAUSAL RELATIONSHIP, Mikovits advocates the use of *not harmless* anti-retrovirals by CFS-patients.
  • At this stage of controversy, the WPI-XMRV test is sold as “a reliable diagnostic tool“ by a firm (VIP Dx) with strong ties to WPI. Mikovits even tells patients in a mail: “First of all the current diagnostic testing will define with essentially 100% accuracy! XMRV infected patients”. WTF!? 
  • This test is not endorsed in Belgium, and even Medicare only reimbursed 15% of the PCR-test.
  • The ties of WPI to RedLabs & VIP Dx are not clearly disclosed in the Science Paper. There is only a small Note (added in proof!)  that Lombardi is operations manager of VIP Dx, “in negotiations with the WPI to offer a diagnostic test for XMRV”.
Please see this earlier post [13] for broader coverage. Or read the post [16] of Keith Grimaldi, scientific director of Eurogene, and expert in personal genomics, who I asked to comment on the “diagnostic” tests. In his post he very clearly describes “what is exactly wrong about selling an unregulated clinical test  to a very vulnerable and exploitable group based on 1 paper on a small isolated sample”.

It is really surprising this wasn’t picked up by the media, by the government or by the scientific community. Will the new findings have any consequences for the XMRV-diagnostic tests? I fear WPI will get away with it for the time being. I agree with Lipkin, who coordinates the NIH-sponsored multi-center CFS-XMRV study that calls to retract the paper are premature at this point . Furthermore, –as addressed by WSJ [17]– if the Science paper is retracted, because XMRV findings are called into question, what about the papers also reporting a  link of XMRV-(like) viruses and CFS or prostate cancer?

WSJ reports, that Schekman, the editor-in chief of 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 findings in a paper don’t hold up: it’s part of the scientific process for different groups to publish findings, for other groups to try to replicate them, and for researchers to debate conflicting results.”

I agree, this is a normal procedure, once the paper is accepted and published. Fraud is a reason to retract a paper, doubt is not.

Notes

*samples, NOT patients, as I saw a patient erroneous interpretation: “if it is contamination in te lab how can I have it as a patient?” (tweet is now deleted). No, according to the contamination -theory” XMRV-contamination is not IN you, but in the processed samples or in the reaction mixtures used.

** The reviewers did ask additional evidence, but not with respect to the PCR-experiments, which are most prone to contamination and false results.

  1. Chronic-Fatigue Paper Is Questioned (online.wsj.com)
  2. 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
  3. WPI Says No to Retraction / Levy Study Dashes Hopes /NCI Shuts the Door on XMR (phoenixrising.me)
  4. http://wpinstitute.org/news/docs/FinalreplytoScienceWPI.pdf
  5. Alberts B. Editorial Expression of Concern. Science. 2011 May 31.
  6. Science asks authors to retract XMRV-chronic fatigue paper; when they refuse, issue Expression of Concern. 2011/05/31/ (retractionwatch.wordpress.com)
  7. K. Knox, Carrigan D, Simmons G, Teque F, Zhou Y, Hackett Jr J, Qiu X, Luk K, Schochetman G, Knox A, Kogelnik AM & Levy JA. No Evidence of Murine-Like Gammaretroviruses in CFS Patients Previously Identified as XMRV-Infected. Science. 2011 May 31. (10.1126/science.1204963).
  8. XMRV and chronic fatigue syndrome: So long, and thanks for all the lulz, Part I [erv] (scienceblogs.com)
  9. Paprotka T, Delviks-Frankenberry KA, Cingoz O, Martinez A, Kung H-J, Tepper CG, Hu W-S , Fivash MJ, Coffin JM, & Pathak VK. Recombinant origin of the retrovirus XMRV. Science. 2011 May 31. (10.1126/science.1205292).
  10. XMRV is a recombinant virus from mice  (Virology Blog : 2011/05/31)
  11. Science asks XMRV authors to retract paper (photoninthedarkness.com : 2011/05/31)
  12. van der Kuyl AC, Berkhout B. XMRV: Not a Mousy Virus. J Formos Med Assoc. 2011 May;110(5):273-4. PDF
  13. Finally a Viral Cause of Chronic Fatigue Syndrome? Or Not? – How Results Can Vary and Depend on Multiple Factor (laikaspoetnik.wordpress.com: 2010/02/15/)
  14. Three Studies Now Refute the Presence of XMRV in Chronic Fatigue Syndrome (CFS) (laikaspoetnik.wordpress.com 2010/04/27)
  15. WPI Announces New, Refined XMRV Culture Test – Available Now Through VIP Dx in Reno (prohealth.com 2010/01/15)
  16. The murky side of physician prescribed LDTs (eurogene.blogspot.com : 2010/09/06)
  17. Given Doubt Cast on CFS-XMRV Link, What About Related Research? (blogs.wsj.com)
  18. Does the NHI/FDA Paper Confirm XMRV in CFS? Well, Ditch the MR and Scratch the X… and… you’ve got MLV. (laikaspoetnik.wordpress.com : 2010/08/30/)

Related articles





Does the NHI/FDA Paper Confirm XMRV in CFS? Well, Ditch the MR and Scratch the X… and… you’ve got MLV.

30 08 2010

ResearchBlogging.orgThe 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.

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

  1. 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
  2. Schekman R (2010). Patients, patience, and the publication process. Proceedings of the National Academy of Sciences of the United States of America PMID: 20798042
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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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Finally a Viral Cause of Chronic Fatigue Syndrome? Or Not? – How Results Can Vary and Depend on Multiple Factors

15 02 2010

Last week @F1000 (on Twitter) alerted me to an interesting discussion at F1000 on  a paper in Science, that linked Chronic fatigue syndrome (CFS) to a newly discovered human virus XRMV [1]ResearchBlogging.org.

This finding was recently disputed by another study in PLOS [2], that couldn’t reproduce the results.  This was highlighted in an excellent post by neuroskeptic “Chronic Fatigue Syndrome in “not caused by single virus” shock!

Here is my take on the discrepancy.

Chronic fatigue syndrome (CFS) is a debilitating disorder with unknown etiology. CFS causes extreme fatigue of the kind that does not go  away after a rest. Symptoms of CFS include fatigue for 6 months or more and experiencing other problems such as muscle pain, memory problems, headaches, pain in multiple joints and  sleep problems. Since other illnesses can cause similar symptoms, CFS is hard to diagnose. (source: Medline Plus).

No one knows what causes CFS, but a viral cause has often been suspected, at least in part of the CFS patients. Because the course of the disease often resembles a post-viral fatigue, CFS has also been referred to as post-viral fatigue syndrome (PVFS).

The article of Lombardi [1], published in October 2009 in Science, was a real breakthrough. The study showed that two thirds of patients with CFS were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV). XMVR was previously linked to prostate cancer.

Lombardi et al  isolated DNA from white blood cells (Peripheral Blood Mononuclear Cells or PBMCs) and assayed the samples for XMRV gag sequences by nested polymerase chain reaction (PCR).

The PCR is a technique that allows the detection of a single or few copies of target DNA by amplifying it across several orders of magnitude, generating thousands to millions of copies of a particular DNA. Nested PCR amplifies the resultant amplicon several orders of magnitude further. In the first round external primers are used (short DNA-sequences that fit the outer end of the piece of DNA to be amplified) and an internal set of primers is used for the second round. Nested PCR is often used if the target DNA is not abundantly present and to avoid the comtamination with products that are amplified as a spin-off due to the amplification of artifacts (sites to which the primers bind as well)

[I used a similar approach 15-20 years ago to identify a lymphoma-characteristic translocation in tonsils and purified B cells of (otherwise) healthy individuals. By direct sequencing I could prove that each sequence was unique in its breakpoint sequence, thereby excluding that the PCR-products arose by contamination of an amplified positive control. All tumor cells had the translocation against one in 100,000 or 1,000,000 normal cells. To be able to detect the oncogene in B cells, B cells had to be purified by FACS. Otherwise the detection limit could not be reached]

Lombardi could detect XMRV gag DNA in 68 of 101 patients (67%) as compared to 8 of 218 (3.7%) healthy controls. Detection of gag as well as env XMRV was confirmed in 7 of 11 CFS samples at the Cleveland Clinic (remarkably these are only shown in Fig 1A of the paper, thus not the original PCR-results).
In contrast, XMRV gag sequences were detected in 8 of 218 (3.7%) PBMC DNA specimens from healthy individuals. Of the 11 healthy control DNA samples analyzed by PCR, only one sample was positive for gag and none for env. The XMRV gag and env sequences were more than 99% similar to those previously reported for prostate tumor–associated strains of XMRV. The authors see this as proof against contamination of samples with prostate cancer associated XMRV-DNA.

Not only PCR experiments were done. Using intracellular flow cytometry and Western blot assays XMRV proteins were found to be expressed in PBMCs from CFS patients. CFS patiens had anti-XMRV antibodies and cell culture experiments revealed that patient-derived XMRV was infectious. These findings are consistent with but do not prove that XMRV may be a contributing factor in the pathogenesis of CFS. XMRV might just be an innocent bystander. However, unlike XMRV-positive prostate cancer cells, XMRV infection status did not not correlate with the RNASEL genotype.

The Erlwein study was published within 3 months after the first article. It is much simpler in design. DNA was extracted from whole blood (not purified white blood cells) and subjected to a nested PCR using another set of primers. The positive control was an end-point dilution of the plasmid. Water served as a negative control. None of the 186 CSF samples was positive.

The question then is: which study is true? (although it should be stressed that the Science paper just shows a link between the virus and CFS, not a causal relationship)

Regional Differences

Both findings could be “real” if there was a regional difference in occurrence of the virus. Indeed XMRV has previously been detected in prostate cancer cells from American patients, but not from German and Irish patients.

Conflict of Interest

Lombardi’s clinic [1] offers $650 diagnostic test to detect XMRV, so it is of real advantage to the authors of the first paper that the CSF-samples are positive for the virus. On the other hand Prof. Simon Wessely of the second paper has built his career on the hypothesis that CFS is a form of psychoneurosis, that should be treated with cognitive behavior therapy. The presence of a viral (biological) cause would not fit in.

Shortcomings of the Lombardi-article [1]

Both studies have used nested PCR to detect XMRV. Because of the enormous amplification potential, PCR can easily lead to contamination (with the positive control) and thus false positive results. Indeed it is very easy to get contamination from an undiluted positive into a weakly positive or negative sample.

Charles Chiu who belongs to the group detecting XMRV in a specific kind of hereditary prostate cancer, puts it like this [5]:

In their Dissenting Opinion of this article, Moore and Shuda raise valid concerns regarding the potential for PCR contamination in this study. Some concerns include 1) the criteria for defining CFS/ME in the patients and in controls were not explicitly defined, 2) nested PCR was used and neither in a blinded nor randomized fashion, 3) the remarkable lack of diversity in the six fully sequenced XMRV genomes (<6 nucleotide average difference across genome) — with Fig. S1 even showing that for one fully sequenced isolate two of the single nucleotide differences were “N’s” — clearly the result of a sequencing error, 4) failure to use Southern blotting to confirm PCR results, and 5) primary nested PCR screening done in one lab as opposed to independent screening from start to finish in two different laboratories. Concerns have also been brought up with respect to the antigen testing

Shortcomings of the Erlwein-article [2]

Many people have objected that the population of CSF patients is not the same in both studies. Sure it is difficult enough to diagnose CSF (which is only done by exclusion), but according to many commenters of the PLOS study there was a clear bias towards more depressed patients. Therefore, a biological agent is less likely the cause of the disease in these patients. In contrast the US patients had all kinds of physical constraints and immunological problems.

The review process was also far less stringent: 3 days versus several months.

The PLOS study might have suffered from the opposite of contamination: failure to amplify the rare CSF-DNA. This is not improbable. The Erlwein group did not purify the blood cells, used other primers, amplified another sequences and did not test DNA of normal individuals. The positive control was diluted in water not in human DNA. The negative control was water.

Omitting cell purification can lead to a lower relative amount of the XMRV-DNA or to inhibition (often seen this with unpurified samples). Furthermore the gel results seem of poor quality (see Fig 2). The second round of the positive PCR sample results in an overloaded lane with too many aspecific bands (lane 9), whereas the first round leads to a very vague low molecular band (lane 10). True that the CSF-samples also run two rounds, but why aren’t the aspecific bands seen here? It would have been better to use a tenfold titration of the positive control in human DNA (this might be a more real imitation of the CSF samples: (possibly) a rare piece of XMRV DNA mixed with genomic DNA) and to use normal DNA as control, not water.Another point is that the normal XMRV-incidence of 1-3,7% in healthy controls is not reached in the PLOS study, although this could be a matter of chance (1 out of 100).

Further Studies

Anyway, we can philosophize, but the answer must await further studies. There are several ongoing efforts.

References

  1. 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
  2. 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
  3. http://f1000biology.com/article/yxfr5q9qnc967kn/id/1166366/evaluation/sections
  4. http://neuroskeptic.blogspot.com/2010/01/chronic-fatigue-syndrome-in-not-caused.html
  5. Charles Chiu: Faculty of 1000 Biology, 19 Jan 2010 http://f1000biology.com/article/id/1166366/evaluation

Photo Credits

Nested PCR ivpresearch.org