It is really hard to take Lombardi et al. 2009 at face value.
The PCR conditions are badly reported in the WPI paper, published in Science. As a matter of fact I wonder how it ever came trough the review.
- Unlike XMRV-positive prostate cancer cells, XMRV infection status did not not correlate with the RNASEL genotype.
- The sensitivity of the PCR is not shown (nor discussed).
- No positive control is mentioned. The negative controls were just vials without added DNA.
- Although the PCR is near the detection limit, only first round products are shown (without confirmation of the identity of the product). The positive bands are really strong, whereas you expect them to be weak (near the detection limit after two rounds). This is suggestive of contamination.
- PBMC have been used as a source and that is fine, but one of WPI’s open letters/news items (Feb 18), in response to the first UK study, says the following:point 7. Perhaps the most important issue to focus on is the low level of XMRV in the blood. XMRV is present in such a small percentage of white blood cells that it is highly unlikely that either UK study’s PCR method could detect it using the methods described. Careful reading of the Science paper shows that increasing the amount of the virus by growing the white blood cells is usually required rather than using white blood cells directly purified from the body. When using PCR alone, the Science authors found that four samples needed to be taken at different times from the same patient in order for XMRV to be detected by PCR in freshly isolated white blood cells.(emphasis mine)
- But carefully reading the methods, mentioned in the “supporting material” I only read:The PBMC (approximately 2 x 107 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 ºC for further culture and analysis or resuspended in TRIzol (…) and stored at -80 ºC for DNA and RNA extraction and analysis. (emphasis mine)Either …. or. Seems clear to me that the PBMC were not cultured for PCR, at least not in the experiments described in the science paper.
How can one accuse other scientists of not “duplicating” the results if the methods are so poorly described and the authors don’t adhere to it themselves??
- Strikingly only those PCR-reactions are shown, performed by the Cleveland Clinic (using one round), not the actual PCR-data performed by WPI. That is really odd.
- It is also not clear whether the results obtained by the various tests were consistent.Suzanne D. Vernon, PhD, Scientific Director of the CFIDS Association of America (charitable organization dedicated to CFS) has digged deeper into the topic. This is what she wrote :So how well are these CFS cases characterized, really?
Of the 101 CFS subjects reported in the paper, results for the various assays are shown for only 32 CFS subjects. Of the 32 CFS subjects whose results for any of the tests are displayed, 12 CFS subjects were positive for XMRV on more than one assay. The other 20 CFS subjects were documented as positive by just one testing method. Using information from a public presentation at the federal CFS Advisory Committee, four of the 12 CFS subjects (WPI 1118, 1150, 1199 and 1125) included in the Science paper were also reported to have cancer – either lymphoma, mantle cell lymphoma or myelodysplasia. The presentation reported that 17 WPI repository CFS subjects with cancer had tested positive for XMRV.
Dr. Mikovits, the gift to the world that keeps on giving.