From PLosOne, 20 February 2012.
Multiple Sources of Contamination in Samples from Patients Reported to Have XMRV Infection
Mary F. Kearney (1*), Jonathan Spindler(1), Ann Wiegand(1), Wei Shao(2), Elizabeth M. Anderson(1), Frank Maldarelli(1), Francis W. Ruscetti(3), John W. Mellors(4), Steve H. Hughes(1), Stuart F. J. Le Grice(1), John M. Coffin(5)
(1) HIV Drug Resistance Program, National Cancer Institute, Frederick, Maryland, United States of America,
(2) Advanced Biomedical Computing Center, SAIC, Frederick, Maryland, United States of America,
(3) Laboratory of Experimental Immunology, Cancer and Inflammation Program, National Cancer Institute, Frederick, Maryland, United States of America,
(4) Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America,
(5) Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts, United States of America
Abstract
Xenotropic murine leukemia virus (MLV)-related retrovirus (XMRV) was reported to be associated with prostate cancer by Urisman, et al. in 2006 and chronic fatigue syndrome (CFS) by Lombardi, et al. in 2009.
To investigate this association, we independently evaluated plasma samples from 4 patients with CFS reported by Lombardi, et al. to have XMRV infection and from 5 healthy controls reported to be XMRV uninfected. We also analyzed viral sequences obtained from supernatants of cell cultures found to contain XMRV after coculture with 9 clinical samples from 8 patients.
A qPCR assay capable of distinguishing XMRV from endogenous MLVs showed that the viral sequences detected in the CFS patient plasma behaved like endogenous MLVs and not XMRV. Single-genome sequences (N = 89) from CFS patient plasma were indistinguishable from endogenous MLVs found in the mouse genome that are distinct from XMRV.
By contrast, XMRV sequences were detected by qPCR in 2 of the 5 plasma samples from healthy controls (sequencing of the qPCR product confirmed XMRV not MLV). Single-genome sequences (N = 234) from the 9 culture supernatants reportedly positive for XMRV were indistinguishable from XMRV sequences obtained from 22Rv1 and XMRV-contaminated 293T cell-lines.
These results indicate that MLV DNA detected in the plasma samples from CFS patients evaluated in this study was from contaminating mouse genomic DNA and that XMRV detected in plasma samples from healthy controls and in cultures of patient samples was due to cross-contamination with XMRV (virus or nucleic acid).
Full paper HERE.
This is Ronald Robert’s take on this paper, posted on Invest in ME’s Facebook page. Thank goodness someone is still prepared to slog through the details. Ron doesn’t mind me reposting his stuff.
John Coffin and his team attempting to prove contamination in the samples used in the Lombardi study once again.
Firstly they decide not to use the samples from the Lombardi study but draw fresh blood samples, then they decide to perform PCR on the plasma of patients who were healthy controls in the Lombardi study, despite no reports of viral sequences being found in the plasma of either ME patients or healthy controls in the Lombardi study.
They could not find any sign of XMRVvp-62 in the patients reporting positive in the lombardi study using an entirely different PCR assay on RNA extracted from PMBCs and not plasma. They seemed to think that this was surprising. They were performing a different PCR assay on a different sample matrix!
They then try and culture the virus using a different cell line to the one used in Lombardi et al (they did use one LnCAP cell of an unknown source though..).
They found that all of their cultures had been contaminated with the virus from the 22rv1 cell line and the 293T cell line which were used in their experiments.
The experiments were done in mouse lab and they are surprised that their samples were contaminated with mouse viruses?
Because their samples were contaminated they want us to believe that the ones in the Lombardi paper were.
The problem they have is that the 22rv1 or 293T cells were not part of the Lombardi experiment and neither the WPI nor the NCI are mouse labs.
If they want to prove contamination in the Lombardi samples why not simply retest the Lombardi samples?
if they start using the scientific method they might even start to enjoy using it!
They also know full well that the gammaretroviruses found by Mikovits and Ruscetti have yet to be sequenced and have nothing to do with XMRV VP62.
Jace – who is Ronald Roberts, please? I’m not sniping, not in the least – just trying to place his commentary in some kind of intellectual framework.
I don’t see how you need much more than reading ability. The contaminates were in the labs who tested the new samples. It says this in the paper! It’s does not take a genius to realise you shouldn’t have those present.
Of the 16 people in this study, five were in Lombardi, but none of the samples were from Lombardi et al. (2009). The title of the article should read, Multiple Sources of Contamination in our Lab in New Samples from Patients Reported to Have MLV-Related Virus Infection Using Different Samples.
“Blood samples were drawn into EDTA-containing tubes by Phlebotomy Services International at the donors homes on January 21, 2010”
It is correct that PCR on plasma did not detect viral sequences in Lombardi et al. So why did these authors test plasma? You can compared the two papers very easily as they are still public access.
These authors don’t even have a full length clone of the virus they say is in 22Rv1 cell line in the Genbank. It is a sequence from two cell lines according to the details, 22Rv1 and CWR-R1. But there are meant to be several proviruses in 22Rv1 cells?
And as for these two viruses dubbed PreXMRV-1 and 2, where is the evidence for those. Where is the evidence for anything else needed for those two suspected viruses to create another virus? Where is the protein evidence? PreXMRV-1 is replication defective. These viruses are more likely, if they both exist, to be decedents of other XMRVs.