Date Published: November 21, 2011
Publisher: Hindawi Publishing Corporation
Author(s): J. Spindler, J. Hackett, X. Qiu, A. Wiegand, V. F. Boltz, P. Swanson, J. H. Bream, L. P. Jacobson, X. Li, C. R. Rinaldo, S. M. Wolinsky, J. M. Coffin, M. F. Kearney, J. W. Mellors.
Xenotropic MLV-Related Virus (XMRV) was recently reported to be associated with prostate cancer and chronic fatigue syndrome (CFS). Infection was also reported in 3.7% of healthy individuals. These highly reported frequencies of infection prompted concerns about the possibility of a new, widespread retroviral epidemic. The Multicenter AIDS Cohort Study (MACS) provides an opportunity to assess the prevalence of XMRV infection and its association with HIV-1 infection among men who have sex with men. Reliable detection of XMRV infection requires the application of multiple diagnostic methods, including detection of human antibodies to XMRV and detection of XMRV nucleic acid. We, therefore, tested 332 patient plasma and PBMC samples obtained from recent visits in a subset of patients in the MACS cohort for XMRV antibodies using Abbott prototype ARCHITECT chemiluminescent immunoassays (CMIAs) and for XMRV RNA and proviral DNA using a XMRV single-copy qPCR assay (X-SCA). Although 9 of 332 (2.7%) samples showed low positive reactivity against a single antigen in the CMIA, none of these samples or matched controls were positive for plasma XMRV RNA or PBMC XMRV DNA by X-SCA. Thus, we found no evidence of XMRV infection among men in the MACS regardless of HIV-1 serostatus.
Xenotropic Murine Leukemia Virus-Related Virus (XMRV) is a recently discovered gammaretrovirus reportedly associated with prostate cancer and chronic fatigue syndrome (CFS) [1, 2]. Urisman et al. first identified XMRV in 2006 in a cohort of prostate cancer patients , followed by Lombardi et al. who reported XMRV infection in 67% of patients with severe CFS and 3.7% of healthy individuals . These initial reports provided a compelling rationale for further investigations into the prevalence of XMRV infection in human populations. However, controversy arose when subsequent studies failed to detect the virus in similar cohorts [3–7]. It was suggested that inconsistencies in detection of XMRV in patient samples could result from varied incidence of infection in different populations, differing criteria for patient selection, and differing detection methods . It was also proposed that virus levels may be chronically low or episodic in patient plasma or tissues, making virus detection difficult . Adding to the complexity, detection of XMRV by PCR is highly susceptible to false positive results due to amplification of closely related endogenous Murine Leukemia Viruses (MLVs) in the mouse genome and the high prevalence of contaminating mouse genomic DNA in many specimens and reagents [9, 10]. Additionally, studies have suggested that XMRV detection is the result of laboratory contamination from infected cell lines [11–14]. Paprotka et al. proposed that XMRV originated as a laboratory artifact when two endogenous mouse proviruses recombined during passaging of a human prostate cancer tumor in nude mice, an event that is highly unlikely to have occurred more than once. The authors, therefore, concluded that published XMRV sequences obtained from patient samples must have come from contamination of samples by virus or DNA from cell lines infected with this recombinant virus . To investigate the human prevalence of XMRV infection, it is clear that reliable detection requires the application of several diagnostic methods used together, including methods that are not influenced by nucleic acid contamination, to avoid reporting potentially high rates of false positives.
The XMRV study by Lombardi et al. published in October 2009 suggested a surprisingly high seroprevalence for XMRV, even among healthy control subjects . Therefore, we set out to evaluate the prevalence of XMRV infection in a possible high-risk cohort. We adopted a multiple diagnostic assay approach to determine the XMRV status of patient samples to minimize the possibility of obtaining false positive results by individual molecular or serologic methods. False positive PCR results may occur from incidental amplification of environmental mouse genomic DNA due to the close relationship between XMRV and mouse proviruses [9, 10]. False positive serology results may occur due to nonspecific or cross-reactive human antibodies . In fact, a recent study reported that ~4% of HTLV-I-infected individuals had antibodies that were cross-reactive to XMRV p15E protein . Therefore, we applied stringent criteria for XMRV positivity aimed at limiting the risk of reporting false positive results. Our criteria for an XMRV infection required that (1) all replicates from X-SCA must be positive, (2) antibodies must be detectable by CMIA and/or Western blot, and (3) nucleic acid and antibody must both be positive. Samples resulting in discordant results from X-SCA replicates were reported as indeterminate. Applying these criteria, we did not identify any samples from the MACS cohort that were consistent with XMRV infection. Nine samples had positive serology, but no detectable XMRV nucleic acid. Although one sample was indeterminate for XMRV RNA by X-SCA, it was antibody negative. XMRV DNA was not detected in any sample tested. Because we did not find any patient to be reactive to multiple XMRV antigens and because all nine patients for whom serum reactivity was observed were negative for XMRV nucleic acid, we concluded that follow-up testing by additional methods, such as virus culture, was not necessary. We believe that the combined approach of sensitive antibody screening and sensitive and specific nucleic acid testing excludes XMRV infection in the cohort studied.