We found no evidence of infection with XMRV among persons with CFS or matched healthy controls from the US by testing with multiple serologic and PCR assays performed independently in three laboratories blinded to the clinical status of the study participants. Our results contrast with the high rate of XMRV detection reported by Lombardi et al.
among both CFS patients and controls, but are in agreement with recent data reported in two large studies in the UK and a smaller study in the Netherlands that could not detect XMRV sequences in CFS patients and one UK study that also failed to detect specific XMRV neutralizing antibody responses in CFS [11
]. Combined, these negative data do not support XMRV as the etiologic agent of the majority of CFS cases.
Several possibilities could explain these discordant results, including technical differences in assays used for the testing in each study. However, the inability of four independent laboratories to replicate the high XMRV prevalence in CFS cases reported by Lombardi et al.
cannot be explained by minor differences in assays used in each study. In addition, testing at CDC utilized the nested XMRV gag
PCR assay used by Lombardi et al.
and Urisman et al.
to identify XMRV infection in CFS and prostate cancer patients, respectively [11
]. Further, to improve assay sensitivity, we used 1 ug of input DNA which is 4-5 times higher than that used by others [11
], all while maintaining an assay sensitivity of 10 copies. To ensure that our testing would not miss genetically diverse XMRV or MuLV strains, we also used a sensitive nested PCR assay with conserved pol
gene primers and found that this testing was also negative confirming the absence of XMRV/MuLV sequences. While PBMC DNA was used in the majority of specimens, 1 ug whole blood DNA was also used in testing 19 CFS cases. This input DNA represents about 350 ng of PBMC DNA which is similar to the amount used by others [11
], thus not affecting the sensitivity of our results. The negative PCR findings were confirmed by an independent laboratory with a second nested gag
PCR assay which provided additional evidence for the absence of XMRV sequences among CFS cases and controls. The primary PCR amplification used in this second test is also that used by Lombardi et al.
which when combined with a nested PCR step has a 3-copy detection threshold.
Antibody responses particularly to Gag and Env proteins are hallmarks of immune responses to retroviral infections including experimental XMRV infection of macaques [22
]. We used a new WB assay to test for anti-XMRV antibodies and showed by using both monoclonal antibodies and polyclonal antisera that this assay detected specifically, and with high titers, reactivity to both XMRV and MuLV Gag and Env proteins. We were unable to detect antibodies to XMRV Gag and Env in any of the CFS and controls specimens by using this WB assay. Likewise, negative results were obtained in a second, independent laboratory by using XMRV-specific ELISA-based and IFA assays. Thus, the observed negative serologic results for all CFS patients reflect an absence of antibody responses and active XMRV infection. Although limited, the negative WB serology observed in 56 healthy controls and 121 blood donors also suggests that the XMRV seroprevalence in this population is not high. Screening of larger numbers of US blood donors using a high throughput ELISA followed by confirmation in a WB test also showed uncommon seropositivity (~0.1%) [22
]. More studies, however, are needed to determine the prevalence of XMRV in healthy populations.
One current limitation of our study, and of others performing serologic and PCR testing for XMRV, is the absence of bona fide positive and negative control specimens from infected and uninfected humans to determine the analytical sensitivity and specificity of the detection assays. Until panels of well-characterized clinical specimens become available, assay validation will be limited to reagents generated experimentally, such as polyclonal and monoclonal antibodies, XMRV plasmids, and XMRV-infected cells.
The selection criteria with which persons with CFS were included in these various studies may also help to explain the incongruent XMRV findings. The study by Lombardi et al.
used samples from the Whittemore Peterson Institute National Tissue Repository
reported to contain specimens from well-characterized cohorts of CFS [11
]. Yet, the paper provides no information regarding the repository or concerning the nature of these cohorts other than that they were collected from private medical practices in several regions of the U.S. where clusters of CFS have been documented [11
]. An absence of details of the CFS cases and controls in this report makes it difficult to replicate and interpret their findings. In contrast, patients in the UK and Netherland studies were typical of CFS patients seen in specialist clinical services in those countries and resemble persons seen in other specialist CFS services in the US and Australia [16
]. Almost half of the UK CFS patients described onset of their illness as related to an acute viral disease [16
]. Thus, they would appear quite comparable to those in the study by Lombardi et al.
Similarly, our study also failed to detect XMRV infection in 18 CFS patients referred to a fatigue registry by health care providers in Georgia and included three persons who reported sudden onset to their illness. Our study is the first to evaluate XMRV infection in persons with CFS and healthy controls from the general populations of Wichita and Georgia. These CFS cases are different from CFS patients seen in general practice and referral clinics; of the participants from the population-based study in Georgia, only half had consulted a physician because of their fatigue, about 16% had been diagnosed with CFS, and 75% described an insidious onset to their illness that had no obvious relation to an acute infectious disease. Nonetheless, results from our general population cohort extend the examination of XMRV in CFS to persons whose illness developed gradually, for the most part, rather than acutely. Our negative findings, in conjunction with those in Europe [16
], indicate no discernable association of XMRV with a wide spectrum of CFS cases. The negative results for CFS patients and controls from the US in the current study also do not support a continental clustering of XMRV infection suggested by the absence of infection in the UK and Netherlands [16
]. However, our findings may not be generalizable beyond our study populations because XMRV infection rates may vary in different regions or locales.
CFS is a diagnosis of exclusion based on self-reported symptoms and requires careful medical and psychiatric evaluations to rule out conditions with similar clinical presentation. Our study and the negative reports from the UK and the Netherlands evaluated patients for exclusionary conditions and defined CFS according to criteria of the 1994 International CFS Research Case Definition [23
] or the earlier Oxford case definition [24
]. The Lombardi et al
. study specifies that samples were selected from patients fulfilling the 1994 international CFS case definition [23
] and the 2003 Canadian Consensus Criteria for CFS/ME [25
]. Lombardi et al.
did not specify if patients were evaluated for exclusionary conditions, or if the study subjects met both definitions, or which patients met either CFS definition. The 1994 International CFS case definition and the Canadian Consensus Criteria are different and do not necessarily identify similar groups of ill persons. Most notably, the Canadian Criteria include multiple abnormal physical findings such as spatial instability, ataxia, muscle weakness and fasciculation, restless leg syndrome, and tender lymphadenopathy. The physical findings in persons meeting the Canadian definition may signal the presence of a neurologic condition considered exclusionary for CFS and thus the XMRV positive persons in the Lombardi et al.
study may represent a clinical subset of patients [11
CFS is a complex disease with various clinical subtypes proposed which could also account for differences in the results obtained in each study [11
]. While there is still no universal agreement on a precise clinical presentation encompassing CFS illness, defining patient characteristics in studies of CFS etiology or pathogenesis remains crucial for making comparisons across various research conclusions.