The History of XMRV

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The retrovirus XMRV.

 

In 2009, the world became interested in the possible association of the retrovirus XMRV with “chronic fatigue syndrome” (CFS). Many studies were conducted on this topic and an enormous amount of news media coverage resulted.

The two research studies that showed an association between XMRV or a similar retrovirus and “CFS” subsequently were retracted from the literature.

The scientific events that led to this occurring are summarized below.

 

The Beginning

On October 8, 2009, the prestigious journal Science released a paper describing the detection of the retrovirus XMRV in the blood cells of patients with “CFS.”

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. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science. 2009 Oct 23; 326 (5952): 585-9. PMID: 19815723

 

The lead author on the paper was Judy Mikovits, at the time employed by the Whittemore Peterson Institute (WPI). (WPI has since changed its name to the Nevada Center for Biomedical Research.)

The laboratories participating in the paper were at the WPI; at the National Cancer Institute (NCI), where Mikovits previously had worked; and at the Cleveland Clinic Foundation.

The paper stated that the authors had found XMRV DNA in 68 of 101 patients with CFS (67%), compared to 8 out of 218 patients (3.7%) in a sample of healthy controls.

XMRV is a murine leukemia virus (MLV) that previously had been reported as being associated with prostate cancer.

Although the term “CFS” was used in the paper, these actually were myalgic encephalomyelitis patients from epidemic outbreaks (including the Lake Tahoe cohort from the mid-1980’s), obtained from WPI’s national tissue repository.

(Note that although physician Daniel L. Peterson has his name on the paper because he helped to obtain patient samples when he was affiliated with the WPI, he left the organization in 2010 and was never involved with the retroviral research project.)

Following is the abstract of the paper published in Science:

Chronic fatigue syndrome (CFS) is a debilitating disease of unknown etiology that is estimated to affect 17 million people worldwide. Studying peripheral blood mononuclear cells (PBMCs) from CFS patients, we identified DNA from a human gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), in 68 of 101 patients (67%) as compared to 8 of 218 (3.7%) healthy controls. Cell culture experiments revealed that patient-derived XMRV is infectious and that both cell-associated and cell-free transmission of the virus are possible. Secondary viral infections were established in uninfected primary lymphocytes and indicator cell lines after their exposure to activated PBMCs, B cells, T cells, or plasma derived from CFS patients. These findings raise the possibility that XMRV may be a contributing factor in the pathogenesis of CFS.

 

Within a month of the release of the paper, the founders/managers of the Whittemore Peterson Institute began offering through private laboratories  (first VIP Dx and then later UNEVX) XMRV tests by culture and serology to the general public, for a cost of up to $650. They continued to offer the tests for almost two years, through the summer of 2011.

Following the publication of the XMRV study in Science, a number of other laboratories looked for XMRV or similar retroviruses in “CFS” patients (defined by various parameters) and published their results.

An FDA laboratory found MLV-related virus gene sequences – though not XMRV in particular – in 67% of a sample of “CFS” patients and in about 4% of healthy controls. The paper was published in the prestigious journal Proceedings of the National Academy of Sciences (PNAS).

Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, Wang R, Alter HJ. Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors. Proc Natl Acad Sci U S A. 2010 Sep 7; 107(36): 15874-9. PMID: 20798047

 

Many other papers reported not finding XMRV or any similar retroviruses in CFS patients, healthy controls or any other populations.

A question thus became whether the laboratories that had reported finding MLV’s in patient populations were reporting a real effect or if those results were not reflective of reality.

 

Blood XMRV Scientific Working Group Paper

The goal of this project was to determine whether the blood supply was at risk of contamination with MLV retroviruses as a result of blood donations by patients suffering from “chronic fatigue syndrome.”

The paper was published online in the journal Science in September 2011 and then later appeared in the printed version of the journal.

Simmons G, Glynn SA, Komaroff AL, Mikovits JA, Tobler LH, Hackett J Jr, Tang N, Switzer WM, Heneine W, Hewlett IK, Zhao J, Lo SC, Alter HJ, Linnen JM, Gao K, Coffin JM, Kearney MF, Ruscetti FW, Pfost MA, Bethel J, Kleinman S, Holmberg JA, Busch MP; Blood XMRV Scientific Research Working Group (SRWG). Failure to confirm XMRV/MLVs in the blood of patients with chronic fatigue syndrome: a multi-laboratory study. Science. 2011 Nov 11;334(6057): 814-7. PMID: 21940862

 

Participating in the project were seven laboratories, each of which analyzed a panel of blinded blood samples to determine whether MLV retroviruses were present in them.

On the panel were representatives from the two papers that had reported finding MLV’s associated with “CFS.” These included Judy A. Mikovits and Max A. Plost of the Whittemore Peterson Institute; Francis W. Ruscetti of the National Cancer Institute; and Shyh-Ching Lo and Harvey J. Alter of the FDA study.

Other participants analyzing samples included Abbott Laboratories; the Centers for Disease Control and Prevention (CDC); Gen-Probe: and a separate FDA laboratory.

The laboratory conducting the original FDA study was referred to in the paper as FDA/Lo. The other FDA laboratory was referred to as FDA/Hewlett.

Each of the seven laboratories was provided with blinded blood samples and asked to test them for the presence of XMRV or other MLV’s. The samples provided were of four different types.

1. Samples from “CFS” patients who had been reported as positive in the original WPI study. (One individual not suffering from “CFS” but who had had close contact with someone with the illness was included in this group.)

2. Samples from “CFS” patients who had been reported as positive in the FDA study.

3. Samples from study controls who had been reported as negative on all tests in either the WPI study or the FDA study.

4. Positive controls – blood samples that had been spiked with XMRV material.

The laboratories used several different kinds of tests to evaluate the samples.

The results are summarized in this table from the paper. They also are discussed below.

 

NAT/Plasma:

The first analysis was a nucleic acid amplification test, looking at plasma.

All of the labs participating in the study stated that 0 out of 10 patients that had been reported as positive in the WPI study were negative on this test.

All of the labs also stated that 0 out of 5 patients that had been reported as positive in the FDA study were negative on this test.

All of the labs except for the WPI stated that 0 out of 15 controls that had been reported as negative in a previous study were positive for MLV’s on this test.

WPI reported that 2 out of the 15 control samples were positive for MLV’s on this test.

All of the labs except for the WPI stated that 5 out of 5 positive spiked controls were positive on this test, demonstrating that they were able to detect XMRV when it was actually present.

WPI reported that only 4 out of 5 of these spiked positive controls were positive on this test.

 

NAT/PBMC:

The next test was another nucleic acid amplification test – this one looking at peripheral blood mononuclear cells (PBMC’s).

All of the laboratories except WPI stated that 0 of 10 patients who were reported as positive in the WPI study were positive on this test.

WPI stated that one of the patients that they had reported as positive in their earlier study was positive on this test.

All of the laboratories stated that 0 out of 10 patients who were reported as positive in the FDA study were positive on this test.

All of the laboratories stated that 0 out of 3 controls that had been reported as negative in a previous study were positive on this test.

All of the laboratories except for the WPI stated that 5 out of 5 positive spiked controls were positive on this test, demonstrating that they were able to detect XMRV when it was actually present.

WPI reported that only 4 out of 5 of these spiked positive controls were positive on this test.

 

NAT/WB:

The next test was a nucleic acid amplification test, using a Western blot.

All of the laboratories reported that 0 out of 10 of the patients who were reported as positive in the WPI study were positive on this test.

All of the laboratories reported that 0 out of 5 of the patients who were reported as positive in the FDA study were positive on this test.

All of the laboratories reported that 0 out of 15 of the controls who were reported as negative in a previous study were positive on this test.

All of the laboratories reported that 5 out of 5 of the spiked positive controls were positive on this test.

 

Serology:

Four of the laboratories evaluated the samples by serology.

Neither Abbott nor the CDC reported finding any evidence of MLV’s in the samples from the WPI patients (0 out of 10), in the samples from the FDA patients (0 out of 5), or in the samples from the negative controls (0 out of 10).

The NCI/Ruscetti laboratory reported finding MLV’s in the samples from the WPI patients (3 out of 10), in the samples from the FDA patients (2 out of 5), and in the negative controls (8 out of 15).

The WPI laboratory reported finding MLV’s in the samples from the WPI patients (5 out of 10), in the samples from the FDA patients (5 out of 5), and in the samples from the negative controls (6 out of 15).

The positive spiked controls were not evaluated in this part of the exercise.

 

Culture:

Three of the laboratories attempted to identify MLV’s in the samples by culture.

The FDA/Hewlett laboratory did not find any MLV using this test in samples from the negative controls (0 out of 15), in samples from the WPI patients that had previously tested positive (0 out of 10), or in samples of the FDA patients that had previously tested positive (0 out of 5). It correctly identified all of the positive spiked controls (5 out of 5).

The NCI laboratory reported that 3 of the 10 WPI patient samples were positive; that 0 out of 5 of the FDA patient samples were positive, and that 6 out of 15 of the negative controls were positive. It correctly identified 5 out of 5 of the positive spiked controls.

WPI stated that their samples had become contaminated with mycoplasma and thus did not submit results of culture for this exercise.

 

Discussion:

Noteworthy in these results is the fact that the only laboratories to report any positives in this study (other than the positive spiked controls) were the two that were involved in the original XMRV paper (WPI and NCI).

These two laboratories also were the only ones participating in the study not to be able to consistently identify the positive spiked controls – thus suggesting that their tests had reliability problems.

With regard to the NAT tests, the paper states:

The only positive NAT results on some of the replicates from clinical samples were reported by the WPI. The WPI assays appeared less sensitive than those used by the other laboratories, based on the fact that only 3 of 5 plasma and 4 of 5 PBMC-spiked positive control replicates were scored as positive by WPI. However, two plasma clinical aliquots were reported as positive in the WPI nested RT-PCR gag assay. These samples were from two different negative controls, and only one out of the three replicates was positive in each case.

 

The paper states about the serology test:

Among all serologic replicates tested, the WPI detected 22 positives, including 10 reactive results among the negative controls, and six each in the subjects previously reported as positive by WPI and by FDA/Lo. Three of the six known negative controls with a positive serology result had at least two of three replicates positive. All five patients previously identified as P-MLV positive by FDA/Lo had a replicate called serology positive, but only one had both replicates reported as positive. Similarly for the 10 subjects previously identified as XMRV positive by WPI, four subjects had one of two replicates reported as serology positive, while both replicates from one patient were reported positive (table 2). There was no significant difference in the rates of positive WPI serology results between negative controls and XMRV/P-MLV cohort subjects (p-value = 0.27). There was no statistical agreement between the samples reported as serology positive by the NCI/Ruscetti and WPI laboratories, despite the fact that they used similar assays.

 

The conclusion of the paper was that the results produced by the WPI and NCI laboratories had demonstrated their tests to be unreliable and that there was no evidence that XMRV or other MLV’s were associated with CFS. The paper states:

In summary, our study demonstrates that no XMRV/P-MLV assay in any of the nine participating laboratories could reproducibly detect XMRV/P-MLV in fifteen subjects (fourteen with CFS) who had previously been reported as XMRV/P-MLV-infected usually at multiple time points and often by multiple assays.

The two laboratories (WPI and NCI/Ruscetti labs) that reported positive results in this study reported similar rates of reactivity among XMRV/P-MLV subjects and known negative control donor samples. The results from both laboratories were inconsistent when their assays were performed in parallel on replicate sample aliquots derived from individual subject specimens.

There was also no agreement of reactivity when comparing results between these two laboratories for the 30 blinded XMRV/P-MLV cohorts and control samples. In contrast, assays developed by FDA (Lo and Hewlett), CDC, NCI/DRP, Abbott Diagnostics, Abbott Molecular and Gen-Probe, all of which have been designed to detect XMRV and relevant MLVs with high sensitivity and specificity, failed to detect evidence of viral infection in any of the previously positive subjects, including CFS patients, or negative control specimens represented in the study.

Based on these findings, we conclude that currently available XMRV/P-MLV assays, including the assays employed by the three participating laboratories that previously reported positive results on samples from CFS patients and controls, cannot reproducibly detect direct virus markers (RNA, DNA, or culture) or specific antibodies in blood samples from subjects previously characterized as XMRV/P-MLV positive (all but one with a diagnosis of CFS) or healthy blood donors. Finally, our findings are reassuring with respect to blood safety and indicate that routine blood donor screening for XMRV/PMLV is not warranted at this time.

 

5-Azacytidine Controversy

The Blood Working Group study was published on September 22, 2011.

Judy Mikovits was fired from her job as research director of the WPI a week later, on September 29.

Also on September 29, patients reported that sales of the XMRV test had been suspended.

The following day, public questions emerged about a figure in the original Science paper presenting the idea that XMRV was related to CFS.

An article in Science Magazine on October 4, 2011, summarized the situation as follows:

The furor revolves around an image—the bottom half of Figure 2C in Lombardi et al.—that shows XMRV proteins in CFS patients but not healthy controls. In her blog known as ERV (endogenous retroviruses), Abbie Smith on 30 September noted the striking similarities between Figure 2C and a slide Mikovits presented at a CFS meeting in Ottawa, Canada, on 23 September. Smith, who is working on her doctoral dissertation at the University of Oklahoma, Oklahoma City, and studies HIV, wrote that an anonymous tipster had pointed out to her that the two images looked identical but had different patient numbers and experimental conditions. Smith questioned whether this was a simple mistake or an attempt to recycle old data to make a new argument.

The Ottawa slide supported Mikovits’s contention that even if XMRV could not be detected in CFS patients, other gammaretroviruses still lurked in their chromosomes. Mikovits described how she had treated cells from two CFS patients with a chemical, 5-azacytidine, that takes methyl groups off DNA. This procedure prods cells that harbor latent versions of retroviruses to produce them, and the image on the slide showed the resultant proteins in what’s known as a Western blot gel. In Lombardi et al. what appears to be the same image shows “XMRV proteins” and makes no mention of 5-azacytidine use.

Mikovits’s collaborator, Francis Ruscetti of the National Cancer Institute (NCI) in Frederick, Maryland, who ran all of the Western blots, confirms that the Ottawa slide uses the same image that appears in Lombardi et al. Ruscetti and Mikovits, in a joint e-mail to Science for this article, said many patients and their doctor, Daniel Peterson (who since has had a falling out with WPI), knew the original coded numbers, so the researchers changed them for the Science publication to “protect the patient privacy.” Ruscetti says it was a mistake for Mikovits to have used the original patient codes in Ottawa. “We were under so much pressure, we missed it,” says Ruscetti.

As far as the use of 5-azacytidine, Ruscetti and Mikovits stressed in their e-mail that “there was no attempt in the original paper to hide anything.” They say for the purposes of Lombardi et al., the use of 5-azacytidine was not germane: They were simply trying to demonstrate that CFS patients had viral proteins not seen in controls. By the time of the Ottawa meeting, they say they realized that this experiment did not in fact show XMRV but proteins from a broader family of gammaretroviruses.

 

Retractions

On December 23, 2011, the editor-in-chief of Science, Bruce Alberts, took the unusual step of retracting the original paper showing an association between XMRV and CFS without the consent of the authors. He wrote:

Science is fully retracting the report “detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome.” Multiple laboratories, including those of the original authors, have failed to reliably detect xenotropic murine leukemia virus–related virus (XMRV) or other murine leukemia virus (MLV )–related viruses in chronic fatigue syndrome (CFS) patients. In addition, there is evidence of poor quality control in a number of specific experiments in the Report. Figure 1, table S1, and fig. S2 have been retracted by the authors.  In response to concerns expressed about Fig. 2C, the authors acknowledged to Science that they omitted important information from the legend of this figure panel. Specifically, they failed to indicate that the CFS patient–derived peripheral blood mononuclear cells (PBMCs) shown in Fig. 2C had been treated with azacytidine as well as phytohemagglutinin and interleukin-2. This was in contrast to the CFS samples shown in Figs. 2A and 2B, which had not been treated with azacytidine.

Given all of these issues, Science has lost confidence in the Report and the validity of its conclusions. We note that the majority of the authors have agreed in principle to retract the Report but they have been unable to agree on the wording of their statement. It is Science’s opinion that a retraction signed by all the authors is unlikely to be forthcoming. We are therefore editorially retracting the Report. We regret the time and resources that the scientific community has devoted to unsuccessful attempts to replicate these results.

 

On January 3, 2012, the authors of the FDA paper published in the Proceedings for the National Academy of Sciences retracted their paper as well.

 

Lipkin Study

This blinded analysis study was stated as having the purpose of putting to rest patient concerns that XMRV might be an issue in the disease. It cost more than a million dollars to run and was spearheaded by the well-known virologist Ian Lipkin of Columbia University.

Alter HJ, Mikovits JA, Switzer WM, Ruscetti FW, Lo SC, Klimas N, Komaroff AL, Montoya JG, Bateman L, Levine S, Peterson D, Levin B, Hanson MR,Genfi A, Bhat M, Zheng H, Wang R, Li B, Hung GC, Lee LL, Sameroff S, Heneine W, Coffin J, Hornig M, Lipkin WI. A multicenter blinded analysis indicates no association between chronic fatigue syndrome/myalgic encephalomyelitis and either xenotropic murine leukemia virus-related virus or polytropic murine leukemia virus. MBio. 2012 Sep 18; 3(5). PMID:22991430

 

The individuals testing samples in this blinded study were Judy A. Mikovits (formerly of the WPI); Francis W. Ruscetti (formerly of the National Cancer Institute); the researchers from the original FDA study that reported the finding of MLV sequences (including Shyh-Ching Lo and Harvey J. Alter); and a team of researchers from the CDC.

All of the authors agreed to support the results of this study rather than challenging them.

The paper described the study’s goals and methodology as follows:

Although many studies failed to replicate the XMRV/pMLV findings, none met the criteria required to rigorously test the association between infection and disease in a multicenter study based on an appropriately powered cohort of well-characterized CFS/ME subjects and matched controls. To unequivocally address this uncertainty, our study engaged the original investigators and laboratories wherein XMRV and pMLV were reported. Prior to initiating the study, all lead investigators at clinical and laboratory sites agreed to the criteria for selecting study subjects and to the strategy for blinding investigators and distributing and analyzing samples. Laboratories used site-specific protocols optimized for molecular or serological detection of XMRV and/or pMLV.

CFS/ME case subjects and controls were recruited using rigorous diagnostic criteria at six sites of excellence in CFS/ME clinical research across the United States. Healthy control subjects frequency matched to CFS/ME subjects by sex, age (within 5 years), race/ethnicity, season at blood sampling, and geographic residence were recruited in Boston, MA; Incline Village, NV; Miami, FL; New York, NY; Palo Alto, CA; and Salt Lake City, UT.

Aliquots of specimens collected from each subject were distributed in duplicate in a blinded fashion to the two teams who initially reported XMRV (Mikovits and Ruscetti) or pMLV (FDA) in CFS populations and to the team that first reported failure to replicate their findings (CDC). To best replicate previous study designs, the FDA and Mikovits/Ruscetti/Hanson labs analyzed subject peripheral blood mononuclear cells (PBMC) and plasma; RNA from cultured cells was sent from the Ruscetti lab to the Hanson lab. The design and interpretation of experiments conducted in the Ruscetti and Hanson labs were guided by Mikovits. The CDC received only subject plasma. Positive controls were also distributed in a blinded fashion to all sites (spiked plasma controls containing 2,000 copies XMRV/ml; spiked PBMC controls containing 200 22Rv1 cells/ml).

 

Although the paper does not provide information on the percentage of positive spiked samples that participants were able to identify correctly, it states:

Testing in the CDC, FDA, and Mikovits/Ruscetti/Hanson laboratories by PCR detected the presence of XMRV and pMLV gene fragments in spiked positive-control samples.

 

None of the laboratories reported any XMRV or other MLV’s in any of the other samples by PCR. Although the team of Mikovits and Ruscetti reported some samples as positive by serology, the percentage (around 6% of samples) was the same in the patient and the negative control group.

According to the paper:

Testing in the CDC, FDA, and Mikovits/Ruscetti/Hanson laboratories by PCR detected the presence of XMRV and pMLV gene fragments in spiked positive-control samples. None of the plasma samples from cases were PCR positive for the presence of XMRV or pMLV at the FDA (n = 121) or CDC (n = 147). None of the plasma samples from controls were PCR positive for XMRV or pMLV at the FDA (n= 110) or CDC (n = 146). None of the uncultured PBMC from cases (n = 121) or controls (111) were PCR positive for XMRV or pMLV at the FDA. PCR testing by the Mikovits/Ruscetti/Hanson group of cultured PBMC from patients (n = 117) and controls (n = 126) was negative for all specimens. The prevalence of plasma antibodies reactive with XMRV in plasma was similar in CFS/ME cases (9 of 147, or 6.1%) and controls (9 of 146, or 6.1%); in the exact Mantel-Haenszel test stratified by site, the P value was 1.0.

 

The paper concluded:

Our results definitively indicate that there is no relationship between CFS/ME and infection with either XMRV or pMLV. Indeed, we did not find any evidence of human infection with XMRV or pMLV in peripheral blood in our sample of 293 subjects. The absence of viral nucleic acid places an upper one-sided 95% confidence limit of 1% for the prevalence in the population sampled. This limit could be an underestimate if the observations were all false negatives. However, even if we suppose the presence of three true positives in 293 samples (1% prevalence) and a detection sensitivity as low as 0.80, the probability that all three true positives would test negative would be 0.008 and the probability that at least one sample would test positive would be 0.992. It is thus extremely unlikely that the failure to find any PCR-positive samples in this study was due to false-negative results. The serology results are more difficult to address given that the assay cannot be validated with plasma from humans with confirmed XMRV or MLV infection. We posit that positive results represent either nonspecific or cross-reactive binding and note that irrespective of explanation, a positive signal does not correlate with case status.

 

-Summary by Lisa Petrison, Ph.D. (2015)

 

 

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