A study published this week provides the most definitive evidence to date disproving previous reports that used molecular testing methods such as qPCR to establish a link between chronic fatigue syndrome, or myalgic encephalomyelitis, and various murine leukemia viruses.
The new study, which involved several researchers and labs that generated the earlier reports, also lends credence to the theory that the findings in those studies were a result of laboratory or reagent contamination coupled with the extreme sensitivity of molecular testing methods.
In addition, the new research lays out a blueprint for future researchers to rigorously test the validity of molecular discoveries, particularly those linking specific pathogens and conditions.
The controversy at hand harkens back to 2006, when scientists first discovered a new gammaretrovirus called xenotropic MLV-related virus, or XMRV, and reported the presence of XMRV sequences in samples from patients with chronic fatigue syndrome or prostate cancer.
A few years later, two separate papers — one published in Science in 2009 and another published in the Proceedings of the National Academy of Sciences in 2010, and both since retracted — fanned the flames of the controversy by reporting the use of molecular detection techniques such as qPCR to detect either XMRV or polytropic murine leukemia virus-related gene sequences in the blood of patients that had been diagnosed with CFS (PCR Insider, 9/2/2010).
Since that time, several other research groups have published studies that used molecular and other testing techniques to debunk an association between CMV and the viruses, and to suggest that laboratory contamination, widespread contamination of commercial qPCR and sample prep reagents with MLV-related sequences, or both likely explained away the erroneous link (PCR Insider 6/9/2011 and 1/5/2012).
Despite these and other studies, "since the index publications clinics have been established for the treatment of [chronic fatigue syndrome] with antiretroviral drugs, and concern has been raised with respect to the safety of the blood supply," wrote the authors of the new study, published this week in mBio, a journal from the American Society for Microbiology.
In addition, the authors wrote, "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 address this, researchers from several academic institutions and government labs — including many of the same scientists and laboratories at the National Institutes of Health and US Centers for Disease Control and Prevention that authored previous studies reporting an association — tested the blood of 147 patients diagnosed with CFS/ME and 146 control patients using similar testing methods employed in previous studies.
More specifically, they used a variety of qPCR-based and related methods, such as quantitative real-time PCR assays for generic pMLV/XMRV pro and gag genes using RNA extracts; and nested PCR assays for MLV gag sequences. They also used similar sample collection and nucleic acid extraction and purification methods, taking extreme measures to avoid potential laboratory contamination. They also used PCR and sample prep products from several vendors, including Promega, Life Technologies' Applied Biosystems unit, Thermo Fisher's Finnzymes business, and Qiagen.
Specimens from each subject were distributed in blinded fashion to the two teams that initially reported XMRV or pMLV in CFS populations, and to the team that first reported a failure to replicate those findings.
None of the labs were able to detect the presence of XMRV or pMLV sequences in the blood samples from patients diagnosed with CMV. Meantime, PCR analysis detected the presence of such genes in spiked positive control samples, but failed to detect the genes in control plasma samples.
The researchers noted that "the absence of viral nucleic acid places an upper one-sided 95 percent confidence limit of 1 percent for the prevalence in the population sampled," making it "extremely unlikely that the failure to find any PCR-positive samples in this study was due to false negative results." The groups also used serological techniques to confirm their findings, conceding that it was difficult to address the possibility of false negatives in these cases because they could not be validated with plasma from humans with confirmed XMRV or MLV infection, but nevertheless adding a layer of confidence to their molecular findings.
"The bottom line is we found no evidence of infection with XMRV and pMLV," Ian Lipkin, a researcher at the Columbia University Mailman School of Public Health at Columbia University and a co-author on the study, said in a statement. "These results refute any correlation between these agents and disease."
In addition, the researchers echoed in their paper the hypotheses generated by several other previous research groups that failed to find a link between CFS and the viruses: that the sensitivity of qPCR and related techniques combined with potential contamination of laboratories and reagents may be a more widespread problem than previously thought.
"Sensitive molecular methods for microbial discovery and surveillance have enabled unique insights into biology and medicine," they wrote in their paper. "However, increased sensitivity for bona fide signal increases the risk that low-level contaminants may also be amplified. This can lead to spurious findings that pose challenges for public health and require an expensive and complex pathogen de-discovery process."
The authors also cited similar pathogen-disease links that were established using various molecular techniques, only to be later disproved, including purported associations between enterovirus 71 and amyotrophic lateral sclerosis or the MMR vaccine and autism.
In the case of CFS/ME and murine leukemia viruses, the authors wrote that "indeed, mouse DNA and murine leukemia virus sequences have been found in commercial reverse transcription PCR reagents. It is imperative, therefore, to establish standardized strategies for rigorously testing the validity of molecular discoveries."