By Suzanne D. Vernon, PhD, Scientific Director and K. Kimberly McCleary, President & CEO
Two papers about xenotropic murine leukemia virus related-virus (XMRV) were published online in Science on May 31, 2011. One describes how XMRV originated (Paprotka et al.) and the other provides more evidence that XMRV and the larger family of xenotropic murine leukemia viruses (X-MLVs) cannot be detected in blood from CFS patients (Knox et al.). Virology professor Vincent Racaniello, PhD has written an excellent summary of the Paprotka et al., paper on Virology Blog, so this article will focus on the study conducted by Konstance Knox, PhD, and her colleagues from five independent institutions.
Study design and methods of Knox et al.
To help readers understand the study design and methods used by Knox et al., we have created a study diagram shown at right and here (in larger format). One of the most important aspects of this paper is that the investigators obtained samples from one of the physicians involved in the original 2009 Science paper linking XMRV and CFS, Lombardi et al., Dr. Daniel Peterson of Sierra Internal Medicine. Several of the papers published after the Lombardi study were plagued by concerns about case criteria used and how patients were selected by clinical collaborators. The involvement of Dr. Peterson in the study led by Knox eliminates case definition and patient selection as confounding variables.
The Knox paper describes two groups of CFS patients called P1 and P2. P1 included 41 CFS patients ranging in age from 5 to 73 years of age. They were sequentially enrolled, meaning that they were 41 CFS patients presenting to Dr. Peterson’s medical practice in a row, and that they were not selected based on meeting specific criteria for certain clinical or laboratory characteristics. As reported in the Knox paper, 37 of these 41 people had been tested for XMRV by the Whittemore Peterson Institute (WPI) or its commercial laboratory, VIP Diagnostics (VIPDx). Of the 37 people who were tested, 26 were told by WPI/VIPDx that they were XMRV-positive. The other 11 were negative. All 41 patients in P1 provided samples that were tested by Wisconsin Viral Research Group (WVRG) for XMRV using nested polymerase chain reaction (PCR) and all 41 were negative. The paper states, “Notably, a chart review of the 41 patients revealed that 19 had two blood samples drawn on the same day by the same phlebotomist, with one sample submitted to VIPDx and the other sent to WVRG.” A comparison of these results showed that VIPDx detected XMRV in 10 of 19, while WVRG did not detect XMRV in any of them (0/19).
This finding led the investigators to collect blood samples from a second group of patients (from the same medical practice) called P2. This group of 29 CFS patients included 26 patients who were selected based on having previous positive XMRV test results from WPI or VIPDx. Nine people were included in both P1 and P2; fresh samples were collected from all 29 subjects in P2. The team used a number of techniques that were based on the original XMRV and X-MLV reports (Lombardi et al. and Lo et al.). They included reverse transcription PCR (RT-PCR) and virus culture; yet again, none of the 29 samples were positive by these methods.
One of the findings from the original Lombardi study that had been addressed in only a few follow-up studies is the report that 9/18 (50 percent) of the samples in that study showed antibody reactivity. Using an assay developed by Abbott laboratories that detects antibodies to XMRV and X-MLVs, 60 plasma samples from P1 and P2 patients were tested. One of the 60 samples was weakly reactive but that result could not be confirmed with further testing, so all samples were considered to be antibody negative. They looked to see if serum from 19 P2 CFS patients and 7 healthy controls could inactivate XMRV and X-MLVs. They found that both viruses could be prevented from infecting cells due to a substance in the human blood, possibly complement proteins. This result confirmed reports from five other groups that suggest it is unlikely that these viruses can establish an infection in human blood.
Further experiments by the Knox team found that 3 of 5 enzymes used for RT-PCR and 9 of 17 other common laboratory reagents contained MLV sequences, creating a possible source for the MLV sequences detected in CFS samples by Lo et al. This finding is similar to published reports from several other groups as noted in the paper.
Although XMRV was not detected in the reagents tested by Knox’s team, they did a final analysis that shows three of the XMRV sequences reported by WPI are 98-100 percent identical to VP62, the specific sequence of XMRV cloned from prostate cancer tissue. WPI has recently submitted short portions of 18 new sequences to GenBank, a public library that houses genetic sequences. A freely available comparison of the WPI’s new XMRV sequences to VP62 shows nearly identical genetic composition. Knox et al. conclude, “Therefore, the remarkable conservation of the WPI-XMRV sequences is most consistent with laboratory contamination with the original infectious VP62.” The clone was used by WPI in its original experiments, as reported in the legend for Figure S3 of the supplemental material to the 2009 Science paper.
Since this paper was published, some have flatly dismissed the findings because the study does not represent an exact replication of the precise methods employed by Lombardi et al. As explained in our article that followed the Journal of Virology study by Shin et al., all investigators working on XMRV have modified their methods to optimize detection of XMRV – including the WPI and VIPDx – and to avoid possible routes of contamination. The replication of a study involves different investigators repeating an experiment using the same methods to determine whether they can arrive at the same result. This is what the Shin study attempted. The Knox study also included repeat testing for XMRV in some of the same CFS samples tested by Lombardi et al. However, how closely the Knox team adhered to the original methods still seems to be a point of fierce debate. As reported by Jon Cohen in Science Now for the journal Science, “‘They didn’t do one thing we did,’ [Mikovits of WPI] says. Levy [senior author of the Knox paper] disagrees, saying, ‘We did it exactly the way they did it.’” In its responses to Science, WPI contends that only Lo et al., represents a replication study, even though it used only PCR to look for X-MLVs. Other experts reviewing all the data disagree with that assessment, particuarly in regard to the Shin and Knox studies. This debate will likely continue.
One way to settle the matter is to complete a multicenter study that includes both the WPI and the Lo et al. investigators.
For that reason and many others, the NIH-sponsored studies on XMRV in CFS should proceed as planned. According to an article in the Washington Post today, NIH director Francis Collins has confirmed that the work will continue. These NIH-sponsored studies include participation from the key investigators and laboratories that originally reported the association of XMRV and X-MLVs with CFS. In addition, these studies involve several clinical collaborators from around the country who are expert in CFS diagnosis and care. In a study being coordinated by “virus hunter” Ian Lipkin at Columbia University, samples collected from the same cohorts will be tested in a head-to-head comparison under blinded conditions using each laboratory’s own protocols. The results will be analyzed and the code broken by Lipkin’s group at Columbia’s Center for Infection and Immunity. No matter what the outcome of these NIH-sponsored studies, this funding has laid the foundation for a national clinical-laboratory network that can provide the impetus for further research on CFS etiology, diagnosis and treatment.
It remains to be seen whether XMRV will provide the answers to better methods of diagnosis and treatment that were heralded in October 2009. There is no doubt that the original report of an association has attracted remarkable scientific talent, increased engagement by NIH and unprecedented awareness of the devastating impact of CFS. No other report among the 5,000+ peer-reviewed articles about CFS has attracted this much attention or such sustained effort to investigate more thoroughly. The debate over XMRV has been polarizing at times, but there is no longer dispute about whether CFS is worthy of scientific endeavor; that, in itself, is progress.
References
Paprotka T, Delviks-Frankenberry KA, Cingoz O, Martinez A, Kung HJ, Tepper CG, Hu WS, Fivash, Jr. MJ, Coffin JM, Pathak VK. Recombination origin of the retrovirus XMRV. Science. 31 May 2011. 10.1126/science.1205292
Knox K, Carrigan D, Simmons G, Teque F, Zhou Y, Hackett, Jr. J, Qui X, Luk KC, Schochetman G, Knox A, Kogelnik AM, Levy JA. No identification of murine-like gammaretroviruses in CFS patients previously identified as XMRV-infected. Science. 2011 May 31:10.1126/science.1204983.
Lombardi VC, Ruscetti FW, Gupta JD, 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. 8 October 2009. 1179052.
Lombardi VC, Ruscetti FW, Gupta JD, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. Supporting online material for Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science. 8 October 2009.
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. Proceedings of the National Academy of Sciences. 23 August 2010. 10.1073/pnas.1006901107
Shin CH, Bateman L, Schlaberg R, Bunker AM, Leonard CJ, Hughen RW, Light AR, Light KC Singh IR. Absence of XMRV and other MLV-related viruses in patients with chronic fatigue syndrome. Journal of Virology, 4 May 2011.
Suzanne Vernon, PhD, is the Association’s scientific director. She has nearly two decades of experience as a microbiologist. K. Kimberly McCleary has served as the Association’s chief staff executive since 1991.
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Thank you for including the definition of replication. This question has been very confusing to me, because no one can seem to agree on what would constitute an attempt to replicate Lombardi, et al. Regardless, I am very relieved that Dr. Francis Collins has reaffirmed his commitment to fund the Lipkin study. All parties seem to think that study will be definitive one way or the other.
The Knox et al. 0/0 study used an assay that couldn’t detect the virus in humans. That’s all that needs to be said.
The XMRV issue won’t be put to rest until the multi-lab studies come out. Would it be possible to ask the CAA if they would be able to do a write-up of the recent Light/Bateman gene expression study, please? So many different topics in that one, such as potential subtypes, importance (necessity?) of exercise challenge, etc. Thank you.
Thanks, John. You’re absolutely right — the Light/Bateman study is an important publication that has promise for improving diagnosis and treatment. We will post a summary of the study here on Research1st and will link here so you’re sure to see it. “Gene expression alterations at baseline and following moderate exercise in patients with CFS and FM.” http://www.ncbi.nlm.nih.gov/pubmed/21615807 This study was funded by the CFIDS Association, NIH and the AFSA.
Here is a detailed summary of the Light/Bateman study, just posted to the Research1st blog: http://www.research1st.com/2011/06/02/exercise-challenge-reveals-potential-cfs-biomarkers/
If the positive findings of Lombardi 2009 are indeed a result of contamination, I have two troubling questions.
Why were patient samples contaminated at a dramatically higher rate than control samples in a blinded study? (Yes, all samples were blinded; see http://www.sciencemag.org/cgi/content/full/328/5980/825-d.)
How did contamination manage to blight exactly the same patient and control samples at the two labs (Cleveland Clinic and the National Cancer Institute) which confirmed the WPI’s results?
I would appreciate hearing Dr. Vernon’s answers to these questions. Thank you.
The question about the very different rates of positives in the Lombardi and Lo studies has come up many times. In the Shin/Singh study, the authors tried to address it. Because it comes up again and again, Suzanne and I noted it in our summary of that study, “The Iteration of X” (http://www.research1st.com/2011/05/05/the-iteration-of-x/).
As for the possibility of contamination in the laboratories of WPI collaborators at Cleveland Clinic and the National Cancer Institute, Dr. Robert Silverman has stated to the Chicago Tribune (March 17, 2011) that “his lab had stored a cell line known to harbor XMRV and he was working to determine if contamination occurred.” (See http://www.chicagotribune.com/health/ct-met-chronic-fatigue-xmrv-20110317,0,6116823.story?page=1)
A recently updated XMRV Q&A on the National Cancer Institute’s website makes the following statement about samples tested there: “a sample of the XMRV viruses reported in the 2009 article has been cultivated from patient samples and was analyzed at NCI. In contrast to the original findings, the new data suggest it is unlikely that these XMRVs were derived from infected patients. Instead, like the other XMRVs that have been sequenced, they appear to be laboratory contaminants.” (http://www.cancer.gov/newscenter/qa/2011/xmrv_qa) We understand that there will be more information from NCI about this later testing.
Thanks as well for the section on replication – an important subject – whose complexity is highlighted by the almost complete disagreement between the two researchers!!!
If these false positives were indeed from laboratory contamination, then that still begs the question: why were healthy controls not testing false positive at the same rate in these contaminated labs? And I wonder what Ms. McCleary’s name adds to what purports to be ‘research news’? Is she now a medical researcher with scientific credentials, or is this just more of the CAA’s political maneuvering to stay in good with the CDC and NIH, rather than with those of us who are ill. Let the Ph.D answer the questions, not the politician.
You do not indicate on the 19 people that were tested by both labs how many were told by WPI that they were positive & how many were negative before the study was done where WPI found 9 positives. The findings somehow prompted a second test P 2 for some reason?
Why would they do a second test?
Carolyn, I could be completely wrong about this, but from what I’ve read/heard about this study the P1 test was an attempted replication of the WPI/XMRV findings and the P2 test was an attempted replication of the Alter/Lo/MLV findings. Again I could be wrong but that’s my (incomplete) understanding of the issue.
The paper states, “Notably, a chart review of the 41 patients revealed that 19 had two blood samples drawn on the same day by the same phlebotomist, with one sample submitted to VIPDx and the other sent to WVRG.” A comparison of these results showed that VIPDx detected XMRV in 10 of 19, while WVRG did not detect XMRV in any of them (0/19). It is not reported whether these 19 patients had been tested earlier by WPI or VIPDx. The conflicting results prompted the researchers to test another group of patients.
Nicely Written, Thumbs up!