Response to Comments on Detection of an Infectious Retrovirus, XMRV, in Blood

[Guest guest]

Cells of Patients with Chronic Fatigue Syndrome "

Judy A. Mikovits1,* and Francis W. Ruscetti2

We reported the detection of the human gammaretrovirus XMRV in 67% of 101

patients with chronic fatigue syndrome (CFS) and in 3.7% of 218 healthy

controls, but we did not claim that XMRV causes CFS. Here, we explain why the

criticisms of Sudlow et al., Lloyd et al., and van der Meer et al. regarding the

selection of patients and controls in our study are unwarranted.

1 Whittemore Institute, Reno, NV 89557, USA.

2 Laboratory of Experimental Immunology, National Cancer Institute–Frederick,

Frederick, MD 21701, USA.

* To whom correspondence should be addressed. E-mail: judym@...

Our study (1) documented the presence of a recently discovered human retrovirus,

XMRV, in a high proportion of patients with chronic fatigue syndrome (CFS) in

comparison with healthy controls. Sudlow et al. (2), Lloyd et al. (3), and van

der Meer et al. (4) raise concerns about the cases and controls described in our

study and thus the validity of our results. First, we wish to emphasize that our

study was not intended to be a detailed clinical description of CFS or an

epidemiological study that would relate particular symptoms, demographics,

duration, pattern of onset, and the like to the presence or viral load of XMRV.

The study was not, nor was it designed to be, a case-control study as Sudlow et

al. (2) imply, for it was the first demonstration of the replication and

production of infectious XMRV in human blood cells. The fact that a number of

the patients tested were from regions of CFS outbreaks does not invalidate the

clinical diagnosis. We hope that our report will stimulate the performance of

many case-control studies that use appropriate virus detection. We certainly

recognize that such studies will be required to determine what role XMRV plays

in the pathogenesis of CFS.

Samples included in our study (1) were from CFS patients who fulfilled both the

Fukuda criteria and the Canadian Consensus Criteria (CCC), regardless of

severity. We regret that a sentence in the original supporting online material

in (1) implied that immunological abnormalities were part of the CFS diagnosis;

indeed, while many such patients do exhibit such abnormalities (5, 6), they were

not required for diagnosis. All patients that met Centers for Disease Control

and Prevention and CCC criteria were accepted; none were excluded. Patient

samples were obtained from 2006 to 2009 and stored in the Whittemore

Institute (WPI) repository. We did not state in Lisbon (7) or elsewhere that the

samples analyzed in (1) were only from patients from documented outbreaks of

CFS, nor did we state that the 101 patients described in (1) exhibited all the

immunological abnormalities described in our Lisbon conference presentation. In

fact, only 25 samples in (1) came from patients identified during the 1984 to

1988 CFS outbreak in Incline Village, Nevada. The remaining 76 samples included

patients with sporadic cases from 12 U.S. states and Canada, including

California, New York, North Carolina, Wisconsin, Michigan, Oregon, New Mexico,

New Jersey, North Dakota, Texas, and Florida. Patients in the study were 67%

female, reflecting the reported gender incidence of CFS, with an age

distribution of 19 to 75 years of age (mean of 55). The healthy control

population, which was similar in age and gender to the patients, was composed of

healthy people who visited doctors' offices in the western United States between

2006 and 2008. The great majority, although not all, of the patients analyzed

were matched in geographic location with controls. As this was not an

epidemiological case-control study, we did not attempt to discern where the

patients believed they contracted CFS; at the time of sample collection, some

were undoubtedly living in an area different from the location where they first

became ill.

The information we provide here and in the accompanying Supporting Online

Material (8) should lay to rest any concerns about " bias " or " confounding. "

Again, the primary aim of the work described in (1) was not to characterize this

clinical condition or to prove a cause for CFS but to demonstrate the existence

of an infectious gammaretrovirus in patients who had been diagnosed with CFS. We

achieved our goal using four different experimental strategies. The original

description of HTLV-1 and HIV-1 involved only one or two patients (9–12),

whereas we detected XMRV in 75 individuals.

We did not state that our study (1) proves the cause of CFS. A large number of

infectious and noninfectious agents have been implicated in CFS, and it is that

fact that makes the puzzle of CFS all the more difficult to solve. At no time

have we wished to raise false hopes among a group of patients who, in general,

have not been treated well by the medical research community. We are aware that

many different pathogens have previously been reported to be associated with CFS

but have not been proven to be causal.

We further note that no cytokine profiles were presented in (1), nor did we

state that abnormal cytokine levels, altered natural killer cell activity, or

particular RNase L profiles were a requirement for inclusion in the study.

Unpublished comments made during a medical conference (7) exploring hypothetical

connections with immune system defects, viral reactivation, and malignancies

should not be used to judge the merits of the science in the published paper.

Regarding the concern raised by Sudlow et al. (2) about potential " expectation

bias, " we point out that the National Cancer Institute (NCI) and the Cleveland

Clinic, whose scientists independently performed experiments and coauthored (1),

were certainly not " established " as laboratories for the purpose of studying

CFS. All samples were blinded, as mandated by the NCI and WPI institutional

review board approvals. All experimental procedures were done by the same

personnel, in the same physical laboratory space, under identical protocols.

Investigators at NCI received 100 samples from individuals without knowing their

health status; furthermore, the samples were sent to NCI directly without

passing through the WPI laboratory space. Laboratory workers at the NCI and the

WPI who performed the polymerase chain reaction (PCR) and immunological studies

used coded, blinded samples that did not reveal the CFS status of the

individuals. The WPI has examined all 218 control and 101 patient samples by

both PCR and serological methods for the presence of XMRV nucleic acid and

antibodies. In addition, NCI used plasma from all 100 samples they received in

infection experiments with LNCaP cells. It was not feasible to examine all 101

patient and 218 control samples with all four XMRV detection methods described

in (1), due to time and resource constraints.

Of the technologies used to identify and isolate XMRV in patients with CFS, PCR

from DNA or cDNA from unstimulated peripheral blood mononuclear cells is the

least sensitive method. We contend that the three recently published negative

PCR studies (13–15) do not qualify as being studies that fail to replicate our

study, as neither the same PCR methodologies were used nor did these studies

draw on the additional cell culture and immunological methods that we employed

to observe XMRV nucleic acids and proteins.

Although we offer to send samples in which we have detected XMRV, the groups

that published these results neither requested nor analyzed any samples we had

found positive for XMRV in our laboratories.

Sudlow et al. erroneously state that we did not consider alternative

explanations for the findings, namely that patients with poor general health may

be more susceptible to viral and other infections. On the contrary, we raised as

a question for future study: " Is XMRV infection a causal factor in the

pathogenesis of CFS or a passenger virus in the immunosuppressed CFS patient

population? " (1). We recognize that the presence of XMRV could be due to

enhanced susceptibility to retroviral infection after development of CFS. A

causal role of XMRV in CFS is an intriguing possibility, given the known

immunosuppressive, neurotropic, and serious consequences of infection with other

known retroviruses.

Supporting Online Material

www.sciencemag.org/cgi/content/full/328/5980/825-d/DC1

SOM Text

References

--------------------------------------------------------------------------------

References and Notes

1. V. C. Lombardi et al., Detection of an infectious retrovirus, XMRV, in blood

cells of patients with chronic fatigue syndrome. Science 326, 585 (2009).

[Abstract/Free Full Text]

2. C. Sudlow, M. Macleod, R. Al-Shahi Salman, J. Stone, Science 328, 825 (2010);

www.sciencemag.org/cgi/content/full/328/5980/825-a. [Abstract/Free Full Text]

3. A. Lloyd, P. White, S. Wessely, M. Sharpe, D. Buchwald, Science 328, 825

(2010); www.sciencemag.org/cgi/content/full/328/5980/825-b. [Abstract/Free Full

Text]

4. J. W. M. van der Meer, M. G. Netea, J. M. D. Galama, F. J. M. van Kuppeveld,

.. Science 328, 825 (2010); www.sciencemag.org/cgi/content/full/328/5980/825-c.

[Abstract/Free Full Text]

5. N. G. Klimas, F. R. Salvato, R. , M. A. Fletcher, Immunologic

abnormalities in chronic fatigue syndrome. J. Clin. Microbiol. 28, 1403 (1990).

[Abstract/Free Full Text]

6. K. J. Maher, N. G. Klimas, M. A. Fletcher, Chronic fatigue syndrome is

associated with diminished intracellular perforin. Clin. Exp. Immunol. 142, 505

(2005). [Web of Science] [Medline]

7. J. A. Mikovits, presentation at Conference on Cellular and Cytokine

Interactions in Health and Disease, Lisbon, Portugal, 17 to 21 October 2009).

8. Additional patient information is provided as Supporting Online Material.

9. F. Barré-Sinoussi et al., Isolation of a T-lymphotropic retrovirus from a

patient at risk for acquired immune deficiency syndrome (AIDS). Science 220, 868

(1983). [Abstract/Free Full Text]

10. R. C. Gallo et al., Isolation of human T-cell leukemia virus in acquired

immune deficiency syndrome (AIDS). Science 220, 865 (1983). [Abstract/Free Full

Text]

11. B. J. Poiesz et al., Detection and isolation of type C retrovirus particles

from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma.

Proc. Natl. Acad. Sci. U.S.A. 77, 7415 (1980). [Abstract/Free Full Text]

12. B. J. Poiesz, F. W. Ruscetti, M. S. Reitz, V. S. Kalyanaraman, R. C. Gallo,

Isolation of a new type C retrovirus (HTLV) in primary uncultured cells of a

patient with Sézary T-cell leukaemia. Nature 294, 268 (1981). [CrossRef]

[Medline]

13. O. Erlwein et al., Failure to detect the novel retrovirus XMRV in chronic

fatigue syndrome. PLoS ONE 5, e8519 (2010). [CrossRef] [Medline]

14. H. C. Groom et al., Absence of xenotropic murine leukaemia virus-related

virus in UK patients with chronic fatigue syndrome. Retrovirology 7, 10 (2010).

[CrossRef] [Medline]

15. F. J. M. van Kuppeveld et al., 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

340, c1018 (2010). [Abstract/Free Full Text]

16. Patent applications were submitted for XMRV detection methods in CFS by the

WPI, a not-for-profit 501c3. J.A.M. has signed over any personal rights she may

have on royalties from these patents to the WPI.

--------------------------------------------------------------------------------

Received for publication 10 November 2009. Accepted for publication 19 April

2010.