Another "Oldies, but Goodies" episode

Thanks to RRM for bringing this to the attention of us all.

Patrick Moore
March 6, 2011, 9:05 pm

Occam’s Razor of Virology: When you find a virus that causes disease, the disease should be easier to understand and not harder.

If you have to come up with more explanations for the virus than you would without it, then you are wrong (e.g., XMRV infection is not a HERV and is restricted to a rare form of prostate cancer and CFS–diseases which share no apparent common features. Somehow, these prostate cancer and CFS patients have a common exposure to this virus as a risk factor despite there being no common epidemiologic patterns. Although XMRV is clearly a murine ERV, people with exposure to mice are not at noticeable risk for prostate cancer or CFS).

It makes more sense that XMRV is a murine ERV that jumped to a human prostate cancer cell line decades ago when it was passaged through a nude mouse as a xenograft (a common practice) as elegantly shown by the Hue et al Retrovirology article. It has since been detected as an intermittent PCR contaminant.

My advice, above all else, is to test samples blindly and randomly. If you have PCR contamination, your results will be “biased toward the mean” of no significant relationship. This is the easiest and least costly confirmation possible. When your postulated virus-disease relationship survives this stringent test, then you possibly have something. Then, the science begins. Randomized and blinded testing has saved me, on multiple occasions, from appearing to be a bigger idiot than my natural talents for idiocy allow.

For what it is worth, when the CFS-XMRV paper was first published, we had severe concerns about its methodology and conclusions, which was published in F1000 (subscription required so it is reprinted below). I think it it is still valid:

Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome.
Lombardi VC, Ruscetti FW, …, Silverman RH, Mikovits JA
Science 2009 Oct 23 326(5952):585 -9 [abstract on PubMed] [full text]
DOI: 10.1126/science.1179052 PMID: 19815723
Competing interests: None declared

Comments

The discovery of the cause of chronic fatigue syndrome would be an extraordinary finding. Rather than providing extraordinary proof, this manuscript has flaws that leave the reader unsure of knowing precisely what was measured.

To detect the xenotropic murine leukemia-like virus (XMRV), the authors used nested-PCR on non-randomized and non-blinded samples, a recipe for uncontrolled PCR contamination. This technique re-amplifies previously cycled products and is inherently prone to intermittent false positivity that has occurred in our lab and many others (e.g. {1} and {2} on which I am the author). This is a concern in light of post-publication claims that XMRV detection rates among chronic fatigue syndrome (CFS) patients have climbed from 67% to 95%, and XMRV tests are now being sold and advertised on the internet at http://www.redlabsusa.com.

Southern blotting, which would allay this suspicion, was not done. Other results in the study also lack support. Flow cytometry and immunostaining with murine leukemia virus (MLV) antibodies were used to directly detect viral proteins in patient cells (see Figure 2A of the paper). The CFS peripheral blood cells have robust monotonic staining rather than the bimodal peaks that are expected from a mixture of infected and uninfected populations of peripheral blood cells. It is not certain whether this level of viremia for an exogenous retrovirus is medically possible. It may, perhaps, be possible but it seems improbable and is a pattern more consistent with a cross-reactive endogenous retroviral antigen.

To confirm this finding, CFS peripheral blood cells (without negative controls in Figure 2B) were immunoblotted using cross-reactive spleen focus-forming virus (SFFV) and MLV antibodies. XMRV gp70 and p30 proteins are found at higher levels in 2 out of 5 CFS peripheral blood samples (1150 and 1221) than in the positive control — HCD-57 cells directly infected with SFFV — a very remarkable result. Repetition with negative control samples (see Figure 2C of the paper) has the higher molecular weight bands cut from the photograph, thus we cannot interpret potential positivity for p30 gag precursor proteins among the control samples (see CFS samples 1199 and 1220 in Figure 2B).

Finally, the positive control HCD-57 cell lane in Figure 2C lane 8 has a completely different banding pattern from the very same control in Figure 2B lane 7 for the p30 gag protein. The elementary issue of whether the authors are measuring XMRV has to be clarified. The fundamental basis for the CFS case and control samples is also not defined at an appropriate level. The samples (supplementary online material) were “selected for this study from patients fulfilling the 1994 CDC Fukuda Criteria for Chronic Fatigue Syndrome (S1) and the 2003 Canadian Consensus Criteria for Chronic Fatigue Syndrome/myalgic encephalomyelitis (CFS/ME) and presenting with severe disability”. These are two separate definitions, the latter published in the “Journal of Chronic Fatigue Syndrome” (which is no longer in print). It is unclear how the samples were selected from these two criteria. No references or cut-offs are given for tests used to clinically define the CFS patients as cases so we are unable to interpret the essential basis for the study. In addition, no description is given to indicate that controls were tested in the same manner as CFS patients; in fact, there is no description for negative control samples at all. For a disease whose diagnosis is controversial, a clear statement of where and how the cases and controls were selected is a critical first step.