Serologic and PCR testing of persons with chronic fatigue syndrome in the United States shows no association with xenotropic or polytropic murine leukemia virus-related viruses
© Satterfield et al; licensee BioMed Central Ltd. 2011
Received: 17 December 2010
Accepted: 22 February 2011
Published: 22 February 2011
In 2009, a newly discovered human retrovirus, xenotropic murine leukemia virus (MuLV)-related virus (XMRV), was reported by Lombardi et al. in 67% of persons from the US with chronic fatigue syndrome (CFS) by PCR detection of gag sequences. Although six subsequent studies have been negative for XMRV, CFS was defined more broadly using only the CDC or Oxford criteria and samples from the US were limited in geographic diversity, both potentially reducing the chances of identifying XMRV positive CFS cases. A seventh study recently found polytropic MuLV sequences, but not XMRV, in a high proportion of persons with CFS. Here we tested blood specimens from 45 CFS cases and 42 persons without CFS from over 20 states in the United States for both XMRV and MuLV. The CFS patients all had a minimum of 6 months of post-exertional malaise and a high degree of disability, the same key symptoms described in the Lombardi et al. study. Using highly sensitive and generic DNA and RNA PCR tests, and a new Western blot assay employing purified whole XMRV as antigen, we found no evidence of XMRV or MuLV in all 45 CFS cases and in the 42 persons without CFS. Our findings, together with previous negative reports, do not suggest an association of XMRV or MuLV in the majority of CFS cases.
The xenotropic murine leukemia virus (MuLV)-related virus (XMRV) is a retrovirus capable of infecting human cell lines and was recently found in some persons with prostate cancer . Conflicting reports of XMRV in Europe and the US show XMRV prevalence between 0 and 27% in prostate cancer patients [2–4]. More recently, Lombardi et al. reported finding XMRV in 67% of persons with chronic fatigue syndrome (CFS) and in 3.6% of healthy controls using PCR, serology, and virus isolation . However, six subsequent studies found no association of XMRV and CFS in the US, Europe and China [6–11]. A more recent study failed to detect XMRV, but found a polytropic MuLV most similar to mouse endogenous retroviruses in 87% of CFS cases .
These discrepant results may be explained by differences in assay sensitivities used in each study, genetic heterogeneity of XMRV, geographic distribution of the virus, or by differences in subgroups of people with CFS. Since PCR assays have become standard tools in research and clinical laboratories, and each study reported using very sensitive assays, it is very unlikely that subtle assay differences contribute to these discordant test results. Some studies also used the same PCR assays as the initial study or generic tests for detecting both XMRV and other variants of MuLV [6–9], supporting further that the negative results were not due to assay differences or the ability to detect divergent viral strains.
The 1994 International Research Case Definition of CFS, currently used by most investigators, acknowledges that CFS subtypes are likely to occur, and encourages investigators to examine criteria to stratify cases, such as by type of onset, gradual or acute . Variations in the approach to case ascertainment as well as in the severity of illness and type of onset could result in different spectrum of illness and potential differences in association with infection or other risk factors. It is also possible that the European studies [6–8] did not find XMRV due to regional differences or that the previous CDC study  was too localized to the regions around Georgia and in Wichita, Kansas. Similarly, a possible geographic clustering of XMRV infection has been observed in prostate cancer patients with most cases occurring in the US [2–4].
Statistics on CFS patients and controls from the U.S
Avg Duration of Illness
Blood samples were shipped from collection centers overnight. Most were processed immediately upon arrival, but a few samples were incubated in the refrigerator for 1 to 2 days prior to separation of the blood components. For component separation, blood was centrifuged and the buffy coat, including the peripheral blood mononuclear cells (PBMCs), was immediately and carefully removed. The buffy coat was either processed immediately or stored at -20°C for later analysis. Nucleic acids were extracted using the Qiagen blood DNA minikit protocol (Qiagen, Valencia, CA). Extracted DNA was quantitated using the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) and checked for integrity with a minimum 260/280 ratio of 1.8 and by ß-actin PCR. Plasma was immediately frozen for later analysis.
PCR oligos and conditions
2794 to 3062
2.5 μg DNA
95°C for 20 s followed by 45 cycles of 95°C for 1 s and 60°C for 20 s 
[6FAM] TGTTCCAGGGGGACT GGCAAGGTACCAccctgg [DABC]2,3
2961 to 3330
1.0 μg DNA
40 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 45 s for both primary and nested PCR 
419 to 1149
0.25 μg DNA; RNA from 62 μL plasma
40 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 45 s for both primary and nested DNA PCR [5, 9]. RT-PCR; Primer 1154R was used for cDNA synthesis at 42°C for 1 hr with the IScript Select cDNA kit (BioRad) followed by 85°C, 5 min to stop the reaction. Nested PCR was then performed as for DNA testing using the Expand High Fidelity PCR System (Roche) and AmpliTaq (Applied Biosystems) for the primary and nested PCRs.
1581 to 1764
RNA from 62 μL plasma
RT-PCR using AgPath-ID one step RT-PCR kit (Applied Biosystems) and BioRad iQ5 iCycler. Reverse primer used for cDNA synthesis at 45°C for 20 min; 95°C for 10 min. 55 cycles at 95°C, 30 s, 52°C, 30 s, 62°C, 30 s.
Absence of XMRV in CFS patients from the U.S
For detection of any new virus, false positive and negative results are always a concern, especially when bona fide positive and negative clinical specimens are not available for assay validation. The PCR tests in this study have been previously shown to detect low levels (≤ 10 copies) of XMRV plasmid in high genomic DNA backgrounds and are capable of generically detecting XMRV and diverse MuLVs [5, 9, 14]. While all the PCR tests used in XMRV studies reported similar sensitivities, it is important to note that each used a different amount of starting DNA. Specifically, the assays of Lo et al. and Lombardi et al. can at best detect 1 copy of XMRV/MuLV in a background of 30 to 50 ng and 100 to 250 ng of DNA respectively [5, 12]. However, in our study, we use the most sensitive PCR test reported to date, with a detection limit of 1 copy of XMRV or MuLV in 2,500 ng of DNA, a 10-83X improved detection limit over the assays used by Lombardi et al. and Lo et al. This indicates that any one of the assays would be able to detect XMRV or MuLV if present in the samples. Moreover, a recent study also demonstrated the importance of using at least 600 ng of input DNA to increase detection of XMRV in prostate cancer patients . XMRV could also be present in blood at levels below the detection limit of PCR, but this seems unlikely given the relatively high frequency of infection reported by Lombardi et al. and Lo et al. in people with CFS using tests with less sensitive PCR tests [5, 12]. Unlike other reports [5, 12], we also found no evidence of active XMRV/MuLV viremia using highly sensitive RT-PCR tests excluding possibilities of peripheral infection seeding the blood compartment from other body locations. Furthermore, WB testing did not detect XMRV or MuLV antibodies in the plasma samples, arguing against the development of an XMRV/MuLV-specific humoral immune response, as is commonly seen with other human retroviral infections, and precluding the possibility of low level viral infection in blood or in other reservoirs. Given the recent finding that an XMRV antibody test, using even a single XMRV protein, had 100% sensitivity for XMRV detection in monkeys after the second week of infection with XMRV, it is highly unlikely that our WB test, which uses purified, whole XMRV as antigen and detects XMRV antibodies in infected macaques, would have missed detecting XMRV infection .
It is also important to note that the report by Lo et al. is not a confirmation of the Lombardi et al. study since like previous studies, this study also failed to identify XMRV in any of the CFS samples or controls [6–12]. Rather, Lo et al. identified a polytropic MuLV sequence in a majority of CFS samples that most closely resembles nonfunctional viruses in mouse genomic DNA, which was confirmed by a truncated Gag sequence in one CFS specimen in their study. Thus, without viral isolation or complete genomes, the infectivity and person-to-person transmissibility of these polytropic viruses are unclear. Others have described the lengthy history and ubiquitous nature of mouse cell or DNA contamination, even in laboratories that have never worked with MuLV's, and concluded that contamination cannot be excluded as a source of the MuLV-like sequences in some studies . Since this report, four laboratories have reported that 100% of polytropic MuLV and/or XMRV sequences found in their CFS and prostate cancer samples stemmed from contamination from commercial reagents and/or other sources [11, 19–21]. In addition, a review on XMRV describes the potential dangers from using polymerases with antibody mediated hot starts, especially those developed from mouse hybridoma cells, such as the Platinum Taq used by Lo et al. . While Lo et al. did not find mouse cell contamination by a retrospective screen of their samples for murine mitochondrial sequences or through the use of numerous water controls, mtDNA screening and water controls are not sufficient to detect the majority of murine genomic DNA contamination [19, 20]. Hue et al. showed that 100% of published XMRV sequences from CFS and prostate cancer samples have less sequence variation than occurs within XMRV in the 22Rv1 cell line, concluding that any discovery of these conserved XMRV sequences in patient samples was due to contamination . Given the high degree of known risk for contamination even in laboratories that have never worked with MuLV's and the historical contamination of human cell lines with MuLVs and other retroviruses [18, 24], it is imperative that murine contamination controls be run in parallel with all human testing. Since both polytropic and xenotropic MuLV's are capable of infecting non-murine cells, other controls will need to be developed to rule out contamination from non-murine sources.
In conclusion, we have used a comprehensive testing strategy, including highly sensitive PCR tests and a novel XMRV WB assay, to show that neither the limited geographic differences of previous studies within the United States nor the condition of post-exertional malaise are the reason for the discordant study results. Further, with what are now seven negative studies, it is highly unlikely that XMRV is present in people with CFS or in control populations as frequently as has been previously reported. The amount of specimen from each of the positive studies has been limiting for independent confirmation of the test results. Thus, different study designs are needed to further investigate an association of XMRV and MuLV in persons with CFS, including carefully defined case control studies in which specimens are collected and processed the same, followed by coded and blinded testing at independent laboratories reporting both detection and absence of infection with these viruses.
This study was sponsored by Cooperative Diagnostics in order to help CFS patients. The authors thank Dr. Robert Silverman at the Cleveland Clinic for the VP62 XMRV plasmid and C7 cell line and Dr. John Hackett at Abbott Diagnostics, Chicago, IL for the XMRV-infected macaque sera. Use of trade names is for identification only and does not imply endorsement by the U.S. Department of Health and Human Services, the Public Health Service, or the Centers for Disease Control and Prevention. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
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