Our analyses of plasma samples independently collected from CFS patients previously reported to be XMRV-infected 
and from healthy controls reported to be XMRV-uninfected 
, and of virus isolation co-culture supernatants identified three different types of sample contamination, leading to false positive detection of XMRV. First, we detected high levels of mouse genomic (both IAP and MLVs) and mouse mitochondrial DNA, but no XMRV sequences, in plasma samples from CFS patients, leading us to conclude that contaminating mouse genomic DNA in this set of plasma samples led to false positive PCR results for XMRV. Second, although the 5 plasma samples from healthy controls were free of mouse DNA, two of them contained contaminating XMRV nucleic acid – most likely plasmid or a PCR amplified DNA product – as shown by the PCR amplification profiles and confirmed by sequencing of the amplified product. Third, our analyses of sequences from viruses reportedly isolated from 8 patients with putative XMRV infection 
revealed that the sequences did not differ among the patients and were indistinguishable from sequences of XMRV in XMRV-infected cell lines indicating that the cultures were cross-contaminated from infected cell lines used in the same laboratory. Specifically, the viruses reportedly isolated from patient samples exhibited very little diversity and were closely related to the 22Rv1
virus, consistent with a virus highly similar or identical to that found in the 22Rv1
cell line after a few cycles of virus replication in culture. These findings indicate that the putative isolations of replicating XMRV from patient samples were likely false positives as a result of cross-contamination of the cultures with XMRV from infected cell lines.
PCR and tissue culture are sensitive methods, and are, as a consequence, susceptible to contamination. Care must be taken both to prevent such contamination and to ensure that the analysis includes proper controls to exclude false positive results. The experimental samples and controls must be collected at the same time, using the identical materials, and processed together under identical conditions. Furthermore, it is important to develop strict criteria for declaring a sample positive, including a requirement that multiple methods should yield positive and negative results for the same samples, and the results should be reproducible. Our independent analyses of samples from patients reported to be XMRV-infected (12), refutes prior evidence of XMRV infection in these patients and argues against XMRV infection of human populations.
Our data show that there are at least three different ways contamination can be misinterpreted as XMRV or MLV infection of humans. The first is mouse DNA, which is a ubiquitous environmental contaminant that can find its way into experimental samples in many different ways. Examples include monoclonal antibodies or other bioproducts prepared in mice or mouse cells, chemicals, disposables, or other materials stored where mice can have access 
, or handling of mouse specimens or cell lines in the same laboratory where human samples are being processed 
. Inbred strains of mice contain around 60 MLV proviruses per haploid genome that can be detected by PCR with an env-
specific probe 
, and some wild subspecies contain even more. Given that approximately 50% of the proviruses may be deleted in the env
region (), one cell may contain over 200 proviruses that can be detected by PCR with gag, pro, or pol
primers, increasing the potential that trace amounts of mouse DNA can give rise to a positive PCR signal. The second source of contamination is cloned or amplified XMRV DNA, including DNA being used as a positive control in diagnostic tests. A microgram of XMRV DNA is approximately 1013
copies. Any laboratory that works with either cloned or amplified XMRV DNA is a potential source of contamination. The third source of contamination is inadvertent spread of XMRV originating from 22Rv1
cells to indicator cells co-cultivated with clinical samples. Although this virus is quite sensitive to human restriction factors such as tetherin and APOBECs 3F and 3G, many established cell lines, like 293T, do not express these factors, and cross-contamination can occur even in laboratories with considerable virology experience, leading to subsequent spread to other cell lines, as was observed for the 293T-XMRV cells reported here. Inadvertent contamination of other human cell lines provides a plausible explanation for XMRV contamination even in laboratories that have never cultured the 22Rv1