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ABBREVIATIONS:
CFS =

chronic fatigue syndrome

DHHS =

Department of Health and Human Services

ME =

myalgic encephalomyelitis

MLV =

murine leukemia virus

NHLBI =

National Heart, Lung, and Blood Institute

SRWG =

Scientific Research Working Group

TMA =

transcription-mediated amplification

WB =

whole blood

WPI =

Whittemore Peterson Institute

XMRV =

xenotropic murine leukemia virus–related virus

Recently, there have been studies that indicate that xenotropic murine leukemia virus (MLV)-related virus (XMRV), a newly described human gammaretrovirus, and other related viruses, may be associated with both prostate cancer and myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS).1-4 It has also been suggested that these viruses have the potential to be transmitted by blood transfusion.5 However, a number of studies have failed to support these associations or indeed detect significant evidence of XMRV in the human population.6-9 Currently, there is insufficient information to determine whether or not XMRV and related viruses are a threat to blood safety. Accordingly, the Department of Health and Human Services (DHHS) has established the Scientific Research Working Group (SRWG) to explore the following questions: What is the prevalence of XMRV in the donor population? Is XMRV transmissible by blood transfusion? And if XMRV is transmissible by transfusion, are there any pathologic consequences for the infected recipient? As a starting point, the SRWG has focused on standardizing the various tests used to detect XMRV in blood samples and has facilitated the sharing of clinical samples between laboratories. This commentary discusses background information relating to blood safety and XMRV and related viruses and outlines the specific actions that the SRWG has taken and plans to take.

IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

XMRV was initially identified in 2006 as a result of a search for an infectious cause or cofactor in prostate cancer. Using a microarray of highly conserved sequences from known viruses, Urisman and colleagues2 detected the presence of a gammaretrovirus within some prostate cancer patients. Upon full sequencing, this virus appeared to be closely related to xenotropic murine leukemia viruses (X-MLV) and was thus termed XMRV.

Subsequent studies using polymerase chain reaction (PCR)-based methods and immunohistochemical staining for XMRV revealed viral nucleic acid or proteins in 6% to 23% of prostate cancers, primarily in malignant epithelial cells from higher-grade tumors.1,10 Controversially, a number of subsequent studies have either failed to detect XMRV entirely in prostate cancer11,12 or have only detected a few cases and at the same prevalence in cancerous and noncancerous prostates.13,14 Thus, the precise association of XMRV with prostate cancer, as well as any direct role in tumorigenesis, remains to be defined.

ME/CFS are poorly defined diseases, currently with no well-accepted cause. Thus, Lombardi and colleagues3 at the Whittemore Peterson Institute (WPI) in Reno, Nevada, launched a study of the association between XMRV and CFS. A total of 67% of patients with CFS were found to be XMRV positive by various PCR techniques.3 Of 57 patients with CFS, 21% were positive for XMRV proviral DNA in unstimulated peripheral blood mononuclear cells (PBMNCs), while 54 and 72% were positive for XMRV RNA in unstimulated and stimulated PBMNCs, respectively,25 Eighty-two percent of patients also had serologic evidence of antibodies cross-reactive to the MLV envelope. Furthermore, nearly 4% of PBMNC samples from normal healthy subjects were also found to harbor XMRV DNA.3 Critically, the finding that XMRV nucleic acid was present in blood was supported by the fact that in 89% of cases the virus could be directly cultured from stimulated PBMNCs and/or patient plasma by coculture with an XMRV-sensitive cell line.3

CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

As noted, a number of laboratories have failed to detect XMRV in prostate cancer patients. Subsequently, with molecular and serologic methods, a series of studies have also failed to detect XMRV in patients with ME/CFS or other populations from the United States, Europe, China, and now Africa.6-9,16-22 What is particularly interesting is the fact that the majority of the studies do not simply fail to detect an association with disease, but rather find little or no evidence of any XMRV infection whatsoever, including in blood donors9,16 and potentially at-risk populations such as patients with HIV/AIDS.6,18,20,22

However, a recent study from Food and Drug Administration (FDA) and National Institutes of Health (NIH) laboratories reported that more than 80% of patients with ME/CFS and nearly 7% of NIH blood donors tested positive using nested PCR of proviral DNA in PBMNCs or whole blood (WB) samples.4 The major difference between these findings and those of Lombardi and colleagues were that the majority of viral DNA amplicons detected in samples in the FDA/NIH study, when partially sequenced, appeared to be more closely related to polytropic MLV (P-MLV) rather than XMRV and thus were simply termed MLV-related viruses, or MRVs. Until full-length virus sequences are obtained and virus isolated, it is not clear whether these represent a group of potential human infectious viruses, whether they be zoonotic infections by polytropic MLVs, or perhaps chimeras between XMRV and P-MRV. Thus far, XMRV is the only “human” gammaretrovirus to be fully sequenced and shown to be infectious in cell lines and animal models2,3,16 and also found to be integrated in human tissue.23

There are numerous possible explanations for why multiple laboratories have published discordant findings on XMRV detection. Patient selection and geographical considerations are likely confounding factors. Likewise, the discovery of other MRVs, in addition to XMRV, may suggest that at least some assays could have lacked sensitivity to detect all potential MRVs in clinical samples. For example, PCR primers or antigens for serologic assays may have been designed to be specific for the initial strains of XMRV that were published2,23 and consequently failed to detect divergent MRVs. Contamination with murine DNA or cells, or other nucleic acid sources such as plasmids, at any stage of the process from sample collection to amplification and detection remains a possibility whenever sensitive methods such as nested PCR are employed. Indeed, a number of recent publications have highlighted the potential for mouse DNA contamination in human samples resulting in the detection of XMRV and other MRV sequences.24-28 However, many laboratories are utilizing sensitive assays for mouse mitochondrial DNA4 and other tests to rule out gross contamination. Also the contamination argument is less convincing for the study by Lombardi and colleagues, which documented culture of infectious virus and serologic responses that correlated with molecular findings. However, caution must be taken in both the description of MRVs as human viruses and their potential association with disease.29 The intricacies of many of the techniques, including sample type, preparation and storage, nucleic acid extraction, use of RNA and/or DNA, amplification method, and detection, are all confounding factors and until multiple laboratories either deploy the same techniques or alternately share large numbers of samples, the exact cause of the discordant results will remain unclear.

A careful study of the literature and published protocols does not reveal any obvious consistent differences between protocols from laboratories that do or do not detect MRVs. For example, an early suggestion was that use of heparin collection tubes3 for blood may introduce mouse material into assays; however, at least one study has detected MRVs in blood collected into ethylenediaminetetraacetate tubes.4 Lombardi and colleagues utilized both genomic and cDNA for detection, often from activated white blood cells (WBCs),3 while many of the negative studies used just genomic DNA isolated directly from purified WBCs,9 followed by PCR without a reverse transcription (RT) step. Thus, it could be supposed that virus was present at low copy number and predominantly as nonintegrated RNA and hence was not detectable with PCR of PBMNCs. However, Lo and coworkers4 used only genomic DNA from freshly purified WBCs or WB and did not employ an RT step, yet detected MRV at high rates. In order to standardize samples and assays and obtain “gold standards” for future studies, the Blood XMRV SRWG has launched a series of collaborative studies to address these questions.

BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

Early in the description of XMRV and ME/CFS, the potential for these gammaretroviruses to be transmitted by blood transfusions or by other blood products was raised,5 leading to the important question—could XMRV pose a threat to the safety of the US blood supply? Faced with the possibility of a potential risk of infectious virus in the blood supply, DHHS facilitated the establishment and coordination of collaborative groups, both internal and external to DHHS, with clearly delineated responsibilities and the necessary expertise in risk assessment, risk management, and risk communication. Thus, in November 2009, the DHHS Blood XMRV SRWG was formed and convened for their first meeting in December. At the same time, the AABB formed an XMRV Interorganizational Task Force (see accompanying commentary in this issue of TRANSFUSION).

The Blood XMRV SRWG is charged with the design and coordination of research studies to evaluate whether XMRV poses a threat to blood safety. The Blood XMRV SRWG is led by the National Heart, Lung, and Blood Institute (NHLBI) and is composed of experts in transfusion medicine, blood banking, retrovirology, and ME/CFS research, as well as representatives from the DHHS, the FDA, the Centers for Disease Control and Prevention (CDC), and the NIH (see roster provided in Appendix A). The group reports to the Public Health Service Blood Organ and Tissue Safety Committee and to the Public Health Service Blood Organ and Tissue Senior Executive Council and liaises with the AABB XMRV Interorganizational Task Force, thus ensuring that the latter is aware of the results of its deliberations and studies.

LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

The SWRG determined that an evaluation of the evidence that transfusion may or may not be associated with the development of ME/CFS and prostate cancer should be undertaken early on because it could potentially impact risk assessment (e.g., research study prioritization) and risk management decisions. The lack of an association between transfusion history and ME/CFS or prostate cancer would be indirect evidence that there does not appear to be transfusion transmission of a causative disease agent. Conversely, the presence of an association with CFS and/or prostate cancer could be indirect evidence for transmission of a causative disease agent by transfusion and would potentially reinforce the possibility that XMRV or MRVs could be transfusion transmitted. Only four relevant reports for prostate cancer and one report for CFS were identified; the findings in these papers are summarized below.

Inoue and colleagues30 followed a cohort of 10,451 Japanese men and women for a mean of 12.8 years, from 1990 to 2003; the cohort included 4401 men who were 40 to 79 years old. Blood transfusion before 1990 was associated with an increase in overall cancer mortality (liver and nonliver cancer; hazard ratio, 1.75; 95% confidence interval [CI], 1.32-2.18) and nonliver cancer mortality (hazard ratio, 1.68; 95% CI, 1.25-2.26). This was secondary to an increase in stomach, liver, and pancreatic cancer mortality but not because of an increase in “other” cancer mortality for men (1.32; 95% CI, 0.67-2.57); the other category would include prostate cancer mortality. Transfusion history was assessed solely by a questionnaire and medical record reviews to corroborate transfusion histories were not performed. These results differed from those obtained in an earlier analysis of the same cohort, which revealed an association between prostate cancer mortality and a blood transfusion history (hazard ratio, 1.71, 95% CI, 1.1-2.66, adjusted for age and area of study).31 The authors did not provide an explanation for these discrepant results. Another study conducted by Blomberg and colleagues32 in Sweden included 28,338 nontransfused patients and 1572 transfused patients followed from 1981 to 1982 until 1991 and used a population-based regional tumor registry to identify incident cancers. They found that there was no excess risk of prostate cancer in either group (there was a trend toward less cancer in the transfused patients than expected but this finding was not significant). Conversely, these investigators found an increase in the incidence of malignant lymphoma and nonmelanoma skin cancer in patients receiving a transfusion compared to patients who did not. Finally, Hjalgrim and coworkers33 used the Scandinavian Donations and Transfusions (SCANDAT) database to evaluate cancer incidence in transfusion recipients followed from 1968 in Sweden and 1982 in Denmark to 2002. Approximately 888,800 transfusion recipients were followed. The standardized incidence ratio for prostate cancer was increased at 1 to 5 months after transfusion (2.64; 95% CI, 2.51-2.77) but not afterward with a standardized incidence ratio of 0.98 at 20 years or more after transfusion. Based on the natural history of prostate cancer, the authors attributed the increased incidence at 1 to 5 months to the clinical recognition at 1 to 5 months of prostate cancers that were subclinical at the time of transfusion. Our interpretation of the data from this large study is that there was no evidence of an association between transfusions and the development of prostate cancer.

The one small study of transfusions and ME/CFS was a case-control study conducted by Bell and colleagues34 involving a cluster of 21 students, 10 to 16 years old, with symptoms of CFS in a farming community in upstate New York (Lyndonville) in 1985; these students were designated as cases. Two controls were then identified and matched for age and sex to each case. None of the cases received a blood transfusion while two of the 42 controls did. Additional to this published study, Vernon and McCleary35 recently conducted a survey to evaluate the proportion of patients with CFS reporting a history of transfusion: 8.1% (124/1529) of patients reported receiving a blood transfusion before their diagnosis, whereas 3.3% (50/1529) reported receiving a transfusion after being diagnosed with CFS. By comparison, Wang and colleagues36 found that 5.3% of 92,581 blood donors surveyed in 1998 and 4.2% of 2.9 million donors giving blood at five geographically disperse US blood centers between 1991 and 2000 reported a history of transfusion.

After a careful review of these data, the Blood XMRV SRWG concluded that there was currently no convincing peer-reviewed evidence that transfusion of blood products was associated with the development of prostate cancer or ME/CFS. However, the group recognized that data were sparse, especially for ME/CFS. Thus, the consensus of the group was that additional epidemiologic studies designed to evaluate the association between transfusion history and ME/CFS or prostate cancer should be conducted if feasible, particularly if XMRV is found to be prevalent in blood donors and transmissible by transfusion. However, it was recognized that conducting rigorous epidemiologic studies with adequate power will be extremely difficult, and the conduct of such protocols was likely outside the mandate and beyond the capacity of the working group.

COORDINATION OF LABORATORY ASSAYS

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

The Blood XMRV SRWG was faced with three basic questions when it first convened in December 2009: Is XMRV in the blood supply, and if so, what is the prevalence of viremia and seroreactivity in donors? Is it transfusion transmitted? If transfusion transmitted, what is the clinical impact on blood recipients? These questions, starting with the simplest one: “What is the prevalence of XMRV in blood donors?” remain unanswered. Lombardi and colleagues3 found evidence of XMRV DNA in eight of 218 (3.7%) PBMNC samples from healthy controls. Similarly, Lo and colleagues4 detected MRV proviral DNA sequences in PBMNC or WB samples from three of 44 (6.8%) healthy NIH blood donors. In contrast, Switzer and coworkers9 found no evidence of proviral DNA in PBMNCs from the 56 healthy controls matched to their CFS cases and 41 blood donors, as well as no serologic evidence by Western blot or enzyme-linked immunoassay of XMRV infection in 53 of the same healthy controls (three had no plasma/serum) or 121 separate blood donors. Although at the time other MRVs had not been described in human samples, it is likely that the assays employed in this study would detect these viruses. Multiple serologic assays employed by Qiu and colleagues16 also detected only inconclusive evidence of XMRV antibody responses in three individuals of more than 1500 blood donations screened. The discordance over prevalence rates in blood donors and similar healthy populations is troublesome, and reaching a consensus on rates of XMRV/MRV infection in donors is a major focus of the SRWG.

The challenge has resided with the identification of sensitive and specific assays that can reliably detect XMRV and MRV nucleic acids and/or immunologic responses to these viruses in blood samples. The discordance described above strongly suggested the need for the development of XMRV analytical reference panels, as well as panels of samples from clinical cases and controls, to be used to validate the performance characteristics of assays for subsequent use to investigate the prevalence of XMRV/MRVs in blood donors and recipients. Thus, the development of XMRV/MRV analytical and clinical panels for characterization of nucleic acid tests (NATs) and serologic assays was identified as a first priority by the Blood XMRV SRWG. Accordingly, a four-phase approach was adopted to achieve these goals, culminating in achieving a consensus for an initial estimate of XMRV/MRV prevalence in US blood donors.

PHASE I: ANALYTICAL SENSITIVITY PANELS

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

As described, because of the large array of different assays employed by researchers, the interpretation of discordant XMRV results has been difficult. Thus, in Phase I of the working group study, which is being funded through the NHLBI Retrovirus Epidemiology Donor Study-II (REDS-II) program, Blood Systems Research Institute, the REDS-II Central Laboratory, developed coded analytical performance panels composed of XMRV virions or XMRV-infected cells serially diluted into plasma or WB, respectively. These panels were designed to allow the participating laboratories to compare XMRV assay sensitivity. Although the majority of studies that have investigated XMRV detection in blood have focused on purified and generally cryopreserved PBMNC preparations, it was decided that for the purposes of blood screening, plasma and WB were much more practical alternative sample types. Panels were prepared using the human prostate cell line, 22Rv1, which harbors at least 10 proviral DNA copies/cell XMRV and produces high titers of infectious XMRV virions in the culture supernatant.37 The approximate XMRV DNA levels in infected cells and RNA viral load in culture supernatant were established by consensus results from three laboratories with quantitative XMRV detection techniques. The diluents for the panels were obtained after several of the participating laboratories prescreened a small number of laboratory volunteers to ensure they were XMRV negative by PCR, serology, and virus culture.

Phase I panels were distributed to five laboratories (CDC, WPI, NCI, and the independent laboratories of Drs Lo and Hewlett at the FDA) involved in the Working Group. In addition, panels were supplied to Gen-Probe, Inc., which although not a part of the SRWG, had recently developed a high-throughput target-capture transcription-mediated amplification (TMA) assay for XMRV on the TIGRIS platform. Thus, their participation was deemed valuable for later stages of the XMRV SRWG research agenda when large numbers of donor and recipient samples would require testing.

Tables 1 and 2 summarize the different sample input levels and methods for nucleic acid extraction, target amplification, and detection employed by the laboratories for WB and plasma, respectively. Three distinct assay types were employed—nested PCR, quantitative real-time PCR, and TMA. Furthermore, a number of the laboratories performed secondary confirmation—either sequencing of product or performance of Southern blots in order to confirm specificity of positive results. Two laboratories employed more than one assay (CDC and FDA [Lo]). For the sake of simplicity in Fig. 1 both laboratories employed the criterion that a positive result in only one assay was sufficient to call a sample positive. Results were reported back to the central laboratory at Blood Systems Research Institute, where the blinded code was broken, and data were analyzed. As seen in Fig. 1, all laboratories detected at least 13 XMRV-infected cells (136 proviral copies/mL), and four of six assays demonstrated even more sensitive limits of detection of 0.5 to 4.5 cells/mL, unfortunately reaching close to the end of the titration series. Four of six plasma RNA assays had limits of detection of 80 or fewer RNA copies/mL; the Gen-Probe assay detected 3.2 or fewer RNA copies/mL.

Table 1. Methods used by participating laboratories for detection of XMRV nucleic acids in WB samples
LaboratoryExtraction methodBlood volume (µL)Input DNA (ng)Assay numberTargetAssay typeQuant/nestedPrimer reference
  • * 

    Forward primer, 5-TGTATCAGTTAACCTACCCGAGT-3′; reverse primer, 5-AGACGGGGGCGGGAAG-3′; XMRV probe, 5′fam-TGGAGTGGCTTTGTTGGGGGACGA-tamra3′.

  • † 

    Gen-Probe employs a duplex TMA assay based on multiple proprietary probes.

  • H = Hewlett; GP = Gen-Probe, Inc.; SCA = single-copy assay.

CDCQIAamp mini blood40010001gagPCR southernNestedSwitzer et al.9
2polPCR southernNestedSwitzer et al.9
FDA (H)QIAamp mini blood200500-1000 gagPCRNestedUrisman et al.2
FDA (Lo)Qiagen DNeasy blood20030-501gagPCRNestedLo et al.4
30-502gagPCRNestedLo et al.4
GPMagnetic-based target capture50NADuplexProprietaryTMANAUnpublished
NCIPromega Wizard genomic DNA5004000 5′ UTR of gagPCR SCAQuantUnpublished*
WPIQIAamp DNA mini blood250100 5′ UTR of gagqPCRQuantDong et al.23
Table 2. Methods used by participating laboratories for detection of XMRV nucleic acids in plasma specimens
LaboratoryExtraction methodVolume plasma (µL)Assay numberTargetAssay typeQuant/nestedPrimer reference
  1. H = Hewlett; GP = Gen-Probe, Inc.; qRT = quantitative reverse transcription; SCA = single-copy assay.

CDCUltracentrifuge/QIAamp viral RNA mini5001proqRT-PCRNestedUnpublished
2gagRT-PCR southernNestedSwitzer et al.9
FDA (H)Qiagen viral RNA mini1401gagRT-PCRNestedUrisman et al.2
FDA (Lo)Ultracentrifuge/TRIzol2501gagRT-PCRNestedLo et al.4
2gagPCRNestedLo et al.4
GPMagnetic-based target capture500DuplexProprietaryTMANAUnpublished
NCIUltracentrifuge including internal standard guanidinium isothiocyanate∼200-500 µL plasma15′ UTR of gagRT-PCR SCAQuantUnpublished
WPIQIAamp viral RNA mini14015′ UTR of gagqRT-PCRQuantDong et al.23
image

Figure 1. Sensitivity of detection of XMRV by multiple laboratories. Analytical panels for WB (A) and plasma (B) were created by serial dilution of 22Rv1 cells or 22Rv1 cell supernatant in WB and plasma, respectively. The dilutions were tested in triplicate by six laboratories (CDC; FDA laboratory of Dr Hewlett [FDA(H)]; FDA laboratory of Dr Lo [FDA(Lo)]; Gen-Probe, Inc., [GP]; NCI; and WPI). Red represents three of three replicates being positive, orange is two of three, yellow is one of three, and white is zero of three. Replicates of six negatives were performed and white represents zero of six, while green represents one of six replicates being positive. In the case of FDA(Lo) and WPI subsequent sequencing demonstrated in each case that the amplification product in the single false-positive result for a negative control sample was of human genomic origin. In the case of GP, a repeat by a separate operator yielded zero of six controls as positive.

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Three negative samples were initially falsely labeled positive—one each in three separate laboratories. However, in the case of two of these results (FDA [Lo] and WPI), subsequent sequencing of the positive bands revealed DNA of human genomic origin, rather than MRV sequences, suggesting nonspecific priming of PCR assays. The third false positive (Gen-Probe) was correctly called negative by the submitting group, because a repeat analysis by a different operator did not detect the sample as positive. Thus, while the number of replicates of negative samples tested was low, little evidence of false positivity was observed. However, these findings do highlight the need for some laboratories to sequence every positive PCR result to confirm specificity. Additionally, the overall similarity of results on the analytical panels suggests that major differences in the sensitivity of assays, at least for the XMRV sequence found in 22Rv1 cells, cannot explain the dramatic differences in XMRV detection in clinical samples reported by the participating laboratories.

PHASE II: PILOT CLINICAL PANELS

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

Although the majority of assays described in Tables 1 and 2, at least in theory, will amplify the polytropic MRVs described by Lo and colleagues4 in addition to XMRV, a major caveat to the Phase I panels is the fact that 22Rv1 cells appear to contain only XMRV, and thus the ability to detect other MRVs, was not directly examined. Consequently, the working group decided that whereas the Phase I study demonstrated the relatively comparable sensitivity of all the assays involved, clinical samples need to be included in the three additional phases (Phases II-IV).

Phase II was designed as a pilot study to provide the participating laboratories their first opportunity to test the same clinical samples, as well as to examine whether the sample types in blood donor repositories collected both specifically for these studies and generally for the study of transfusion-transmitted infections38 were suitable for XMRV detection. These repositories consist primarily of plasma and/or serum and, in some instances, frozen WB aliquots. The SRWG also wanted to ensure that the routine delay between blood collection and sample processing would not adversely affect XMRV detection in clinical samples. Blood was collected under current institutional review board approval from four patients with CFS found to be XMRV positive in the 2009 WPI study published in Science,3 as well as a XMRV/MRV-negative controls identified in Phase I. Specimen tubes were then divided into three groups, with one set immediately processed into PBMNCs, WB, and plasma, while the other two sets were refrigerated and then similarly processed after 2 and 4 days. These samples were coded, blinded, and distributed to three SRWG laboratories—the CDC, the NCI, and WPI, as well as to Gen-Probe. Phase II is currently in progress. The findings will be used to optimize the development of the clinical sensitivity and specificity panels for Phase III (see below) and will inform sample preparation for future studies under development, such as a new NIH study aimed at further evaluating the association between XMRV/MRVs and ME/CFS.39

PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

Phase III will develop larger clinical panels to assess the ability of participant assays to effectively detect XMRV/MRVs in clinical specimens that will be processed by methods optimized in Phase II, that is, a choice will be made between using PBMNCs or WB for cellular samples, and whether samples should be processed immediately after phlebotomy (Day 0) or at 1 to 4 days. To attempt to overcome the issues of variation in viral sequences as well as possible sources of nonspecificity, the Phase III panels will include relatively larger numbers of pedigreed XMRV-positive and -negative clinical samples. These will include samples from approximately 30 patients with ME/CFS reported as positive for XMRV/MRVs by PCR, serology, and/or virus culture in the studies by Lombardi and colleagues and Lo and colleagues;3,4 thus both XMRV- and PMRV-positive clinical samples will be represented. For negative specimens, samples prepared from 10 additional pedigreed XMRV/MRV-negative donors will be included to introduce human host genetic variability—these donors have been prescreened as negative by 1) PCR at the WPI, FDA (Lo), CDC, and Gen-Probe laboratories; 2) serology at the WPI, CDC, and NCI (Ruscetti) laboratories; and 3) culture at the NCI (Ruscetti) laboratory. These positive and negative samples will be assembled into coded panels and distributed to all participating laboratories for XMRV/MRV NAT. In addition, samples for serologic analyses will also be included, allowing an initial indication of sensitivity and specificity of serologic assays developed by some of the participating laboratories and the correlation between detection of XMRV/MRV antibodies and nucleic acids.

PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

Phase IV will be composed of blinded panels of WB and plasma collected from approximately 300 blood donors. Additionally, samples that are known to be XMRV/MRV positive and negative will be included. Blinded panels will be distributed to at least four of the participating laboratories for testing. Test results obtained using WB will be compared to those obtained using plasma for each laboratory; results will also be compared between laboratories. Also, the proportion of donors who are positive for XMRV/MRV DNA, RNA, and/or antibodies (XMRV/MRV prevalence) will be estimated based on compiled results from each laboratory. In addition to providing preliminary information as to the prevalence of XMRV/MRVs in blood donors, it is hoped that this series of studies will yield protocols that can be the basis of a set of “gold standard” assays allowing the standardization of tests across laboratories to minimalize the type of controversies thus far seen in XMRV/MRV detection.

FUTURE STUDIES FOR POTENTIAL CONSIDERATION

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

Depending on the results of Phase IV, additional studies may need to be considered. For example, it likely will be necessary to further evaluate donor prevalence in a larger and more geographically dispersed donor population using both molecular and serologic methods. If the samples stored in the NHLBI donor and donor-recipient repositories are deemed adequate for testing (i.e., if sample preparation and cryopreservation methods are not an issue) studies to assess the temporal distribution of XMRV/MRV in blood donors may be warranted; such studies could be done by accessing various NHLBI repositories that span the past four decades.38 Phase IV results may also help define the feasibility, sample size, and power of studies to assess infectivity by transfusion. A large enough XMRV/MRV prevalence in blood donors would ensure sufficient statistical power to investigate whether XMRV/MRVs are transfusion transmissible using samples from the linked NHLBI donor-recipient repositories (and estimate rates of transmission by XMRV-MLV–reactive donations). Further, if XMRV/MRVs are found to be transmissible by transfusion, studies can be conducted to achieve a greater understanding of the correlates of transmission and of the viral and immunologic variables of acute infection. This in turn would help define the optimal approach to blood screening if the latter was deemed necessary and in particular would highlight the relative value of serologic versus NAT screening.

CONCLUSIONS

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

Over the past two decades, there has been a focus within the blood banking community not only on emerging infectious diseases but also on the emerging knowledge of known infectious agents and the risk they may pose to transfusion recipients.40 Evaluating emerging infectious agents that may affect the safety of the blood supply includes early recognition of potential threats and development of scientific priorities, as well as having a proactive, flexible, and funded strategic research agenda that will effectively and rapidly mobilize available expertise and resources to address pressing questions in a scientifically sound and timely manner. In this commentary we have presented our methodic approach to address the current scientific knowledge gaps in XMRV/MRV and through the NHLBI-funded REDS-II program have been responsive by developing and coordinating multilaboratory studies of XMRV/MRV related to the safety of the nation's blood supply. The creation and combined efforts of the Blood XMRV SRWG and the AABB Interorganizational Task Force are critical to addressing the possibility that XMRV/MRV may turn out to be transfusion-transmitted pathogen(s). We hope that this investigation will not only help evaluate whether XMRV/MRV represent a threat to the safety of the nation's blood supply, but also contribute new science related to this potentially important class of retroviruses.

ACKNOWLEDGMENTS

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

The authors acknowledge the tremendous effort contributed by all SRWG members, as well as the AABB and many laboratory members from all of the contributing laboratories, including Leslie Tobler, Ingrid Wilson, Brian Custer, and Lubov Pitina at Blood Systems Research Institute; HaoQiang Zheng, Hongwei Jia, Shaohua Tang, and Anupama Shankar at CDC; Max Pfost, Cassandra Puccinelli, and Kathyrn Hagen at WPI; Kui Gao at Gen-Probe, Inc.; Frank Ruscetti, Mary Kearney, and Rachel Bagni at NCI; and Shixing Tang and Jiangqin Zhao in OBRR, FDA. The laboratory work was funded by the NHLBI REDS-II Central Laboratory Contract to Blood Systems Research Institute (N01 HB-57181). 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 or the Department of Health and Human Services.

REFERENCES

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix
  • 1
    Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR. XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors. Proc Natl Acad Sci U S A 2009;106:16351-6.
  • 2
    Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, Malathi K, Magi-Galluzzi C, Tubbs RR, Ganem D, Silverman RH, DeRisi JL. Identification of a novel gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog 2006;2:e25.
  • 3
    Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 2009;326:585-9.
  • 4
    Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, Wang R, Alter HJ. Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors. Proc Natl Acad Sci U S A 2010;107:15874-9.
  • 5
    Coffin JM, Stoye JP. Virology. A new virus for old diseases? Science 2009;326:530-1.
  • 6
    Barnes E, Flanagan P, Brown A, Robinson N, Brown H, McClure M, Oxenius A, Collier J, Weber J, Günthard HF, Hirschel B, Fidler S, Phillips R, Frater J. Failure to detect xenotropic murine leukemia virus-related virus in blood of individuals at high risk of blood-borne viral infections. J Infect Dis 2010;202:1482-5.
  • 7
    Erlwein O, Kaye S, McClure MO, Weber J, Wills G, Collier D, Wessely S, Cleare A. Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome. PLoS ONE 2010;5:e8519.
  • 8
    Groom HC, Boucherit VC, Makinson K, Randal E, Baptista S, Hagan S, Gow JW, Mattes FM, Breuer J, Kerr JR, Stoye JP, Bishop KN. Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome. Retrovirology 2010;7:10.
  • 9
    Switzer WM, Jia H, Hohn O, Zheng H, Tang S, Shankar A, Bannert N, Simmons G, Hendry RM, Falkenberg VR, Reeves WC, Heneine W. Absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States. Retrovirology 2010;7:57.
  • 10
    Danielson BP, Ayala GE, Kimata JT. Detection of xenotropic murine leukemia virus-related virus in normal and tumor tissue of patients from the southern United States with prostate cancer is dependent on specific polymerase chain reaction conditions. J Infect Dis 2010;202:1470-7.
  • 11
    Hohn O, Krause H, Barbarotto P, Niederstadt L, Beimforde N, Denner J, Miller K, Kurth R, Bannert N. Lack of evidence for xenotropic murine leukemia virus-related virus (XMRV) in German prostate cancer patients. Retrovirology 2009;6:92.
  • 12
    Aloia AL, Sfanos KS, Isaacs WB, Zheng Q, Maldarelli F, De Marzo AM, Rein A. XMRV: a new virus in prostate cancer? Cancer Res 2010;70:10028-33.
  • 13
    Fischer N, Hellwinkel O, Schulz C, Chun FK, Huland H, Aepfelbacher M, Schlomm T. Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer. J Clin Virol 2008;43:277-83.
  • 14
    Verhaegh GW, de Jong AS, Smit FP, Jannink SA, Melchers WJ, Schalken JA. Prevalence of human xenotropic murine leukemia virus-related gammaretrovirus (XMRV) in Dutch prostate cancer patients. Prostate 2010. Epub Sep 28.
  • 15
    Mikovits JA, Lombardi VC, Pfost MA, Hagen KS, Ruscetti FW. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Virulence 2010;1:386-90.
  • 16
    Qiu X, Swanson P, Luk KC, Tu B, Villinger F, Das Gupta J, Silverman RH, Klein EA, Devare S, Schochetman G, Hackett J Jr. Characterization of antibodies elicited by XMRV infection and development of immunoassays useful for epidemiologic studies. Retrovirology 2010;7:68.
  • 17
    Henrich TJ, Li JZ, Felsenstein D, Kotton CN, Plenge RM, Pereyra F, Marty FM, Lin NH, Grazioso P, Crochiere DM, Eggers D, Kuritzkes DR, Tsibris AM. Xenotropic murine leukemia virus-related virus prevalence in patients with chronic fatigue syndrome or chronic immunomodulatory conditions. J Infect Dis 2010;202:1478-81.
  • 18
    Kunstman KJ, Bhattacharya T, Flaherty J, Phair JP, Wolinsky SM. Absence of xenotropic murine leukemia virus-related virus in blood cells of men at risk for and infected with HIV. AIDS 2010;24:1784-5.
  • 19
    van Kuppeveld FJ, de Jong AS, Lanke KH, Verhaegh GW, Melchers WJ, Swanink CM, Bleijenberg G, Netea MG, Galama JM, van der Meer JW. Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort. BMJ 2010;340:c1018.
  • 20
    Cornelissen M, Zorgdrager F, Blom P, Jurriaans S, Repping S, van Leeuwen E, Bakker M, Berkhout B, van der Kuyl AC. Lack of detection of XMRV in seminal plasma from HIV-1 infected men in The Netherlands. PLoS ONE 2010;5:e12040.
  • 21
    Hong P, Li J, Li Y. Failure to detect xenotropic murine leukaemia virus-related virus in Chinese patients with chronic fatigue syndrome. Virol J 2010;7:224.
  • 22
    Tang S, Zhao J, Viswanath R, Nyambi PN, Redd AD, Dastyar A, Spacek LA, Quinn TC, Wang X, Wood O, Gaddam D, Devadas K, Hewlett IK. Absence of detectable xenotropic murine leukemia virus-related virus in plasma or peripheral blood mononuclear cells of human immunodeficiency virus Type 1—infected blood donors or individuals in Africa. Transfusion 2011;51:463-8.
  • 23
    Dong B, Kim S, Hong S, Das Gupta J, Malathi K, Klein EA, Ganem D, Derisi JL, Chow SA, Silverman RH. An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors. Proc Natl Acad Sci U S A 2007;104:1655-60.
  • 24
    Hue S, Gray ER, Gall A, Katzourakis A, Tan CP, Houldcroft CJ, McLaren S, Pillay D, Futreal A, Garson JA, Pybus OG, Kellam P, Towers GJ. Disease-associated XMRV sequences are consistent with laboratory contamination. Retrovirology 2010;7:111.
  • 25
    Oakes B, Tai AK, Cingoz O, Henefield MH, Levine S, Coffin JM, Huber BT. Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences. Retrovirology 2010;7:109.
  • 26
    Robinson MJ, Erlwein OW, Kaye S, Weber J, Cingoz O, Patel A, Walker MM, Kim WJ, Uiprasertkul M, Coffin JM, McClure MO. Mouse DNA contamination in human tissue tested for XMRV. Retrovirology 2010;7:108.
  • 27
    Sato E, Furuta RA, Miyazawa T. An endogenous murine leukemia viral genome contaminant in a commercial RT-PCR kit is amplified using standard primers for XMRV. Retrovirology 2010;7:110.
  • 28
    Smith RA. Contamination of clinical specimens with MLV-encoding nucleic acids: implications for XMRV and other candidate human retroviruses. Retrovirology 2010;7:112.
  • 29
    Weiss RA. A cautionary tale of virus and disease. BMC Biol 2010;8:124.
  • 30
    Inoue Y, Wada Y, Motohashi Y, Koizumi A. History of blood transfusion before 1990 is associated with increased risk for cancer mortality independently of liver disease: a prospective long-term follow-up study. Environ Health Prev Med 2009. Epub Dec 17.
  • 31
    Kikuchi S. Personal past history and mortality in the Japan Collaborative Cohort Study for Evaluation of Cancer (JACC). Asian Pac J Cancer Prev 2007;8(Suppl):9-20.
  • 32
    Blomberg J, Moller T, Olsson H, Anderson H, Jonsson M. Cancer morbidity in blood recipients—results of a cohort study. Eur J Cancer 1993;29A:2101-5.
  • 33
    Hjalgrim H, Edgren G, Rostgaard K, Reilly M, Tran TN, Titlestad KE, Shanwell A, Jersild C, Adami J, Wikman A, Gridley G, Wideroff L, Nyrén O, Melbye M. Cancer incidence in blood transfusion recipients. J Natl Cancer Inst 2007;99:1864-74.
  • 34
    Bell KM, Cookfair D, Bell DS, Reese P, Cooper L. Risk factors associated with chronic fatigue syndrome in a cluster of pediatric cases. Rev Infect Dis 1991;13(Suppl 1):S32-8.
  • 35
    Vernon SD, McKleary KK. Blood donation and transfusion in CFS patients [abstract]. Rev Antivir Ther Infect Dis 2010;8:30.
  • 36
    Wang B, Higgins MJ, Kleinman S, Schreiber GB, Murphy EL, Glynn SA, Wright DJ, Nass CC, Chang D, Busch MP; Retrovirus Epidemiology Donor Study. Comparison of demographic and donation profiles and transfusion-transmissible disease markers and risk rates in previously transfused and nontransfused blood donors. Transfusion 2004;44:1243-51.
  • 37
    Knouf EC, Metzger MJ, Mitchell PS, Arroyo JD, Chevillet JR, Tewari M, Miller AD. Multiple integrated copies and high-level production of the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) from 22Rv1 prostate carcinoma cells. J Virol 2009;83:7353-6.
  • 38
    Busch MP, Glynn SA. Use of blood-donor and transfusion-recipient biospecimen repositories to address emerging blood-safety concerns and advance infectious disease research: the National Heart, Lung, and Blood Institute Biologic Specimen Repository. J Infect Dis 2009;199:1564-6.
  • 39
    Enserink M. New XMRV paper looks good, skeptics admit—yet doubts linger. Science 2010;329:1000.
  • 40
    Stramer SL, Hollinger FB, Katz LM, Kleinman S, Metzel PS, Gregory KR, Dodd RY. Emerging infectious disease agents and their potential threat to transfusion safety. Transfusion 2009;49(Suppl 2):1S-29S.

Appendix

  1. Top of page
  2. IDENTIFICATION AND POTENTIAL DISEASE CORRELATES OF XMRV
  3. CONTROVERSIES IN XMRV DETECTION IN ME/CFS PATIENTS AND NORMAL DONORS
  4. BLOOD SAFETY CONCERNS LEADING TO FORMATION OF THE BLOOD XMRV SRWG
  5. LACK OF EVIDENCE FOR AN ASSOCIATION BETWEEN TRANSFUSION AND ME/CFS OR PROSTATE CANCER
  6. COORDINATION OF LABORATORY ASSAYS
  7. PHASE I: ANALYTICAL SENSITIVITY PANELS
  8. PHASE II: PILOT CLINICAL PANELS
  9. PHASE III: CLINICAL SENSITIVITY AND SPECIFICITY PANELS
  10. PHASE IV: CLINICAL PANEL FOR DONOR PREVALENCE
  11. FUTURE STUDIES FOR POTENTIAL CONSIDERATION
  12. CONCLUSIONS
  13. ACKNOWLEDGMENTS
  14. CONFLICT OF INTEREST
  15. REFERENCES
  16. Appendix

APPENDIX A

Roster for the Blood XMRV SRWG

Simone Glynn, MD, MSc, MPH

Chair

Branch Chief, Transfusion Medicine and Cellular Therapeutics Branch

Division of Blood Diseases and Resources, National Heart, Lung, and Blood Institute

Jerry A. Holmberg, PhD

Co-Chair

Senior Advisor for Blood Policy

Executive Secretary of the Advisory Committee on Blood Safety and Availability

U.S. Department of Health and Human Services, Office of the Secretary

Office of Public Health and Science

Celso Bianco, MD

Executive Vice President

America's Blood Centers

Michael P. Busch, MD, PhD

Director, Blood Systems Research Institute

Vice President, Research/Scientific Affairs, Blood Systems Professor of Laboratory Medicine, UCSF

Roger Y. Dodd, PhD

Vice President, Research and Development

American Red Cross, Holland Laboratory

Louis M. Katz, MD

Executive Vice President

Medical Affairs, Mississippi Valley Regional Blood Center

Steven H. Kleinman, MD

AABB Liaison

Clinical Professor of Pathology, University of British Columbia

Anthony L. Komaroff, MD

The Simcox-Clifford-Higby Professor of Medicine

Harvard Medical School

Senior Physician, Brigham & Women's Hospital

Judy A. Mikovits, PhD

Research Director, Whittemore Peterson Institute

Department Microbiology and Immunology Applied Research Facility

Graham Simmons, PhD

Associate Investigator

Blood Systems Research Institute

Department of Laboratory Medicine

University of California, San Francisco

Susan L. Stramer, PhD

Executive Scientific Officer

American Red Cross, Scientific Support Office

Leslie H. Tobler, DrPH

Senior Scientist

Blood Systems Research Institute

Suzanne D. Vernon, PhD

Scientific Director

The CFIDS Association of America

NIH

Harvey Alter, MD

Clinical Center

National Institutes of Health

John Coffin, PhD

Contractor

NIH/NCI/DBS/HDRP/RRL

National Institutes of Health

Tufts University, Boston

Dennis F. Mangan, PhD

Chair, Trans-NIH ME/CFS Research Working Group

Senior Research Advisor, Office of Research on Women's Health

Office of the Director

National Institutes of Health

Francis Ruscetti, PhD

Senior Investigator

NIH/NCI/DBS/CIP/LEI

National Institutes of Health

CDC

William Bower, MD

Medical Epidemiologist

Centers for Disease Control and Prevention

R. Michael Hendry, DSc

Chief, Laboratory Branch

Division of HIV/AIDS Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention

Centers for Disease Control and Prevention

Walid Heneine, PhD

Laboratory Branch,

Division of HIV/AIDS Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention

Centers for Disease Control and Prevention

Stephan S. Monroe, PhD

Director of the CDC Division of Viral and Rickettsial Diseases

Centers for Disease Control and Prevention

William Switzer, MPH

Laboratory Branch,

Division of HIV/AIDS Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention

Centers for Disease Control and Prevention

FDA

Jay Epstein, MD

Director

Office of Blood Research and Review

Center for Biologics Evaluation and Research

Food and Drug Administration

Indira Hewlett, PhD

Chief, Laboratory of Molecular Virology

Office of Blood Research and Review

Center for Biologics Evaluation and Research

Food and Drug Administration

Shyh-Ching Lo, MD, PhD

Director, Tissue Safety Program Division of Cellular and Gene Therapies & Division of Human Tissues

Center for Biologics Evaluation and Research

Food and Drug Administration