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PCR Testing Casts Doubt on XMRV Link to Chronic Fatigue Syndrome; Reveals Reagent Contamination

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By Ben Butkus

A pair of recently published research papers used PCR testing to provide perhaps the strongest evidence to date debunking previous reports that detected gene sequences from a virus related to murine leukemia virus in patients diagnosed with chronic fatigue syndrome, suggesting a causative relationship between the two.

In addition, the new studies suggest that laboratory contamination was the likely cause of previous evidence linking the murine viruses to CFS, underscoring the idea that researchers conducting PCR experiments should be extremely cautious about both contaminated commercial reagents and contamination from laboratory manipulation, one of the study's authors said this week.

In the first study, published last week in Science, a team led by researchers from the Wisconsin Viral Research Group, the Open Medicine Institute, and the University of California, San Francisco, among other academic institutions, used PCR testing to evaluate blood samples from 61 patients diagnosed with CFS from a single clinical practice, 43 of whom had previously been identified as positive for xenotropic murine leukemia virus-related virus, or XMRV, and found no evidence of XMRV or other MLVs in the samples.

The study, which was supported in part by Abbott Laboratories, also found that MLV sequences could be detected in commercial laboratory reagents; and determined that the gammaretroviruses in question were strongly susceptible to inactivation by sera from CFS patients and healthy controls, suggesting that an MLV infection in humans would be unlikely.

In the second study, published late last month in PLoS One, a team led by scientists from the UK's National Transfusion Microbiology Laboratories of the National Health Service Blood and Transplant unit used PCR testing to investigate 80 random blood donors who had previously tested negative for XMRV by two other PCR-based techniques.

Despite the expectation that the tests would be negative, they were able to generate XMRV gag PCR signals from two of the samples. Suspecting that the PCR master mixes were the source of the contamination, they tested 88 replicates of water using only Invitrogen Platinum Taq and Applied Biosystems Taq Gold LD master mixes, and were able to generate four XMRV gag sequences with the Invitrogen reagents; and an additional sequence by re-amplifying a first-round product using the ABI product.

The UK researchers concluded that "methodologies for the detection of viruses highly homologous to endogenous murine viruses require special caution as the very reagents used in the detection process can be a source of contamination and at a level where it is not immediately apparent. It is suggested that such contamination is likely to explain the apparent presence of [polytropic] MLV in CFS." More succinctly, they summarized their findings in the title of their paper: "PCR Master Mixes Harbour Murine DNA Sequences. Caveat Emptor!"

The controversy surrounding whether the murine leukemia virus-related viruses, most notably XMRV, are linked to CFS took shape in 2009 when a team led by researchers from the Whittemore Peterson Institute published a paper in Science that used PCR-based testing to find that a high percentage of patients diagnosed with CFS were infected with XMRV. Gene sequences from the same virus had also previously been reported to have been identified in samples of human prostate cancer tissue about four years prior.

However, two subsequent studies failed to find an infectious MLV-related virus in prostate cancer patients from Germany; and four other studies conducted in Europe and the US failed when trying to use PCR to detect XMRV or MLV-related viral gene sequences in the blood of CFS patients.

Then, in August of last year, another study published in the Proceedings of the National Academy of Science fanned the flames of the controversy once again. In that study, a group led by researchers from the US Food and Drug Administration and National Institutes of Health used nested PCR assays to detect MLV-like virus gag gene sequences in patients diagnosed with chronic fatigue syndrome.

The FDA and NIH researchers chalked up the discordance of their results to differences in sample collection, processing methods, and PCR protocols and primers; and presented strong evidence that their own PCR methods were airtight and free of potential contamination (PCR Insider, 9/2/2010).

However, the two most recently published paper — particularly the Science publication — provide some of the strongest evidence to date that XMRV and related murine-like viruses are not associated with CFS, and that previous reports linking the two were most likely the result of laboratory contamination.

"It's now clear — you either get contamination from mouse reagents, or you have a cell line that had XMRV in your lab, and the virus or the cells got into your experiment," Jay Levy, a professor of medicine, research associate in the Cancer Research Institute at UCSF, and corresponding author on the Science paper, told PCR Insider this week.

"This is a strictly mouse virus," he added. "It's not associated, as far as I'm concerned, with … prostate cancer — they're going to find out it's a contaminant there, too — and it's not in CFS. It's not in the human blood supply. And really, we've spent a lot of time and money because of these [previous] reports, and they've been completely debunked by not only our paper, which I think was very definitive, but many others."

According to Levy, contamination of this sort is a pervasive problem in PCR experiments due to the extreme sensitivity of the technique in amplifying trace amounts of genetic material.

"It's routine," Levy said. "Some people will tell you they take showers before they go into reactions. Now they have labs where you prepare the reagents in one room and undertake the reaction in another room. You've got to be very careful to control for small amounts [of DNA], or you can get misled, as what's happened here."

Levy also noted that his group's results were particularly convincing because they tested patients from the same cohort that had been examined in the original Science paper from 2009. Many other studies attempting to disprove a link between murine-like viruses and CFS had been criticized for not testing samples from patients that had previously tested positive.

"Here we have the same clinical practice, we have the same doctor sending them to us, and we even had 43 of the 61 who were already told they were XMRV-positive, and they tested nine of them more than once," Levy said. "So I think we covered it all."

Levy said that Daniel Peterson, the clinical researcher at the Whittemore Peterson Institute who co-authored the first Science paper, "was really the hero in this" as the doctor that provided the UCSF team with the patient samples for their study.

The group believes that it has closed the book on the murine-like virus-CFS connection, and will not pursue further research to determine whether human manipulation or commercial reagents are the most likely causes of contamination.

Levy said that manufacturers of commercial PCR reagents may begin to more carefully screen for murine-like virus genetic material in their products, although "I don't know if they have to, because many times you're just not going to look for a mouse virus."

Nevertheless, he added, researchers in the future need to be more vigilant about the possibility of both contaminated reagents and laboratory contamination.

"You just don't have anything in your lab that's going to contaminate," he said. In their study, he added, "we had the … xenotropic mouse virus … in separate rooms in separate incubators. So we made sure that there was no chance of contamination that way. Although we're usually very careful, anyway — if you know you have to avoid contamination, you're not going to get it."

The researchers disclosed in their Science paper that Abbott, whose researchers also participated in the study, have filed patent applications related to the detection of XMRV using immunoassays and molecular-based assays.


Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.

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