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Analysis of cerebrospinal fluid from chronic fatigue syndrome patients for multiple human ubiquitous viruses and xenotropic murine leukemia-related virus.

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RAPID COMMUNICATION
Analysis of Cerebrospinal
Fluid from Chronic Fatigue
Syndrome Patients for
Multiple Human Ubiquitous
Viruses
and Xenotropic Murine
Leukemia-Related Virus
Steven E. Schutzer, MD,1 Megan A. Rounds, MS,3
Benjamin H. Natelson, MD,1,2,4
David J. Ecker, PhD,3 and Mark W. Eshoo, PhD3
Recent reports showed many patients with chronic fatigue syndrome (CFS) harbor a retrovirus, xenotropic murine leukemia-related virus (XMRV), in blood; other
studies could not replicate this finding. A useful next step
would be to examine cerebrospinal fluid, because in
some patients CFS is thought to be a brain disorder.
Finding a microbe in the central nervous system would
have greater significance than in blood because of the integrity of the blood–brain barrier. We examined cerebrospinal fluid from 43 CFS patients using polymerase chain
reaction techniques, but did not find XMRV or multiple
other common viruses, suggesting that exploration of
other causes or pathogenetic mechanisms is warranted.
cating that the syndrome is, at least for some patients, a
CNS disease.7 CSF is important because it is an accessible
liquid window on the brain. In contrast to finding
microbes in the peripheral blood system, identifying a particular microbe in the CNS of several patients with a similar condition would have greater etiologic significance
because of the inherent integrity of the blood–brain barrier.
Our focus in this study was on XMRV. However,
we also analyzed the CSF for other common viruses to
increase confidence that if we did find XMRV, it was not
among a plethora of other viruses, which could weaken
the argument for pathogenic association. As a first step
to potentially linking XMRV to the neurologic manifestations of CFS, we examined a sequentially collected set
of CSF samples from patients who fulfilled the 1994
case definition for CFS.1 We used 2 different polymerase
chain reaction (PCR)-based techniques to probe for the
presence of XMRV and other common viruses in these
CSF samples. The first PCR method was a broad range
technique, used as a viral screen to probe for the presence
of multiple common human viruses. It did not require a
priori knowledge of what the target microbe could be.8
The second was a traditional targeted PCR method,
where the primers were identical to those of the initial
publication,2 identifying the XMRV retrovirus in the
blood of CFS individuals.
ANN NEUROL 2011;69:735–738
Patients and Methods
C
hronic fatigue syndrome (CFS) remains a medically
unexplained condition.1 Infectious causes have been
suspected, but none has been proven as the cause.
Recently 2 studies have reported that a majority of CFS
patients harbor a gammaretrovirus, xenotropic murine
leukemia-related virus (XMRV), in blood.2,3 This finding
has raised speculation that infection with this retrovirus
may be a cause of the syndrome. However, 4 other
laboratories could not replicate this finding, and 4 new
studies support its presence, but as a laboratory contaminant.4–6 Distinctions need to be made between association of a microbe and its causality in a disease. Further
distinctions need to be made between even a bona fide
microbial association and the actual pathogenesis of a disease. The central nervous system (CNS) and cerebrospinal
fluid (CSF), which have potential to provide important
insights, have not been included in these investigations.
We believe CSF from CFS patients may be a very
appropriate site to examine because of substantial data indi-
CSF Specimens
CFS SUBJECTS. The patients, aged 18 to 54 years (median
¼ 43 years), all fulfilled the 1994 case definition for CFS1 after
undergoing a careful history and physical examination by medical staff expert in evaluating patients with medically unexplained fatigue and pain. Patients had blood tests to rule out
common causes of severe fatigue such as anemia, liver disease,
From the Departments of 1Medicine, 2Neurology, University of Medicine
and Dentistry of New Jersey–New Jersey Medical School, Newark, NJ;
3
Ibis Biosciences, Inc., Carlsbad, CA; 4Albert Einstein School of Medicine,
Bronx, NY.
Address correspondence to Schutzer, UMDNJ-New Jersey Medical
School, 185 South Orange Ave, Newark, NJ 07103.
E-mail: schutzer@umdnj.edu
Received Dec 22, 2010, and in revised form Jan 20, 2011. Accepted for
publication Jan 24, 2011.
View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.
22389
C 2011 American Neurological Association
V
735
ANNALS
of Neurology
hypothyroidism, systemic lupus erythematosus, and Lyme disease.9 All subjects then underwent a psychiatric diagnostic interview (Diagnostic Interview Schedule for the Diagnostic and Statistical Manual of Mental Disorders, 4th edition) designed to
identify major psychiatric diagnoses and exclude patients with
these diagnoses from further study. Subjects then underwent
lumbar puncture with institutional review board-approved
informed consent. CSF was sent to the laboratory for white
blood cell count (WBC), total protein, and cytokine assays. A
majority of CFS patients had normal CSF protein and cell
counts (protein <45mg/dl and WBC 5/mm3). Ten of the
patients had increased protein values ranging from 46 to 93mg/
dl, with a median of 59mg/dl, and 3 patients had minimally
elevated WBC counts of 6, 7, and 9, respectively.
Broad Range PCR/Electrospray Ionization Mass
Spectrometry Method
Total nucleic acids (DNA and RNA) were isolated from 100ll
aliquots of CSF, and the extracted nucleic acids were suspended
in 100ll of elution buffer using a previously described Qiagen
protocol,10 but without the bead-beating, as this was CSF.
Methods for broad range virus detection, screening for other
viruses, were conducted using PCR/electrospray ionization mass
spectrometry (ESI-MS) as described previously.8 The broad
viral assay used was the Ibis Sterile Fluids Viral assay consisting
of primer pairs targeted to human adenoviruses,11 alphaviruses,12 herpes viruses (human herpesvirus [HHV] 1, 2, 3, 4, 5,
8), human parvovirus B19, dengue viruses 1 to 4, West Nile virus (WNV), Japanese encephalitis virus (JEV), systemic lupus
erythematosus (SLE), enteroviruses A to D, and
coxsackieviruses.
XMRV Specific PCR Method
The XMRV cDNA was tested in a dilution-to-extinction experiment. Using the same kit used in the Lombardi et al paper,2
cDNA was synthesized from a random 10 samples, and then
the gag and env primer pairs were used to amplify the cDNA
with the same buffer and cycling conditions used by Lombardi
et al2 (reverse transcriptase [RT]-PCR). PCR products were
resolved by electrophoresis (3ll sample loaded) on 1% agarose
gels and visualized using a DigiDoc-It Darkroom UV cabinet
(UVP, Upland, CA).
Repeat Specific PCR
We replicated the VIP Dx and protocol as described in Lombardi et al.2 CSF total nucleic acid aliquots from 43 CFS
patients were pooled into 2 groups of equal total volume and
controls, and placed in individual analytical tubes. Tube 1 contained nucleic acid material from the CSF from the first 23
patients. Tube 2 contained nucleic acid material from the CSF
from the remaining 20 patients and 3 negative control extracts.
Tubes 3 and 4 contained distilled water alone. Tube 5 contained distilled water spiked with 100 genome copies of cDNA
of XMRV, which represented at least 2 the limit of our detection in optimization experiments. These tubes provided the calculated necessary volume for specific PCR reactions (gag and
736
env primers) and for cocultivation/cell line transformation
assays. These were provided in a blinded fashion to the VIP Dx
laboratory. This was done to reduce the possibility of interlaboratory differences despite using the same primers, buffers,
and running cycles as described by Lombardi et al.2
Cocultivation of Material from CSF Samples
with Permissive Reporter Cell Line
We used VIP Dx in the capacity of a service laboratory. The VIP Dx
XMRV PCR/culture test is a 2-stage process. First, clinical material
(cells or fluid) are probed by PCR directly for XMRV.2 Second and
independently, the clinical sample is cocultivated with a cell line
(LNCaP) permissive for XMRV infection. After weeks of culture or
visible transformation, the exposed cell line is probed by PCR for
XMRV. The same cell line exposed to known XMRV-negative
human material serves as a negative control to be assayed in the
same fashion. The same permissive LNCaP cell line, not exposed to
any potential XMRV human cells or fluid, can also serve as a negative control. Positive controls are their in-house native controls, previously found to be XMRV positive. To maintain the integrity of a
mutually agreed upon blinded process, our samples were submitted
to the VIP Dx laboratory for full processing in their assay.
Results
Broad Viral Detection by RT-PCR/ESI Assay
All 43 individual CSF samples (based on available volumes) from CFS patients analyzed with the broad range
RT-PCR/ESI-MS8 were negative for human adenoviruses,
alpha viruses, herpes viruses (HHV 1, 2, 3, 4, 5, 8),
human parvovirus B19, dengue viruses 1 to 4, WNV,
JEV, SLE, enteroviruses A to D, and coxsackieviruses.
Limit of detection LOD95 experiments for these viruses
in RT-PCR/ESI-MS were determined in genome copy
number compared to internally calibrated competitive
PCR, and ranged from 15 to 125 genome copies per
PCR reaction as follows: human adenovirus, 15; HHV
1, 30; HHV 4, 60; HHV 5, 30; coxsackievirus, 30; dengue, 60; human parvovirus B19, 125. The genome copy
numbers of the viral stocks determined by internally calibrated competitive PCR were shown to be within 10fold of real time PCR reactions for each individual virus
stock. In the present experiment, 100ll of CSF was
extracted to yield 100ll of eluted volume, from which
3ll was put into each PCR reaction.
Specific PCR
The cDNA was amplified with the gag primer pair down
to 13 copies/ll (1:1,000 dilution) and with the env
primer pair down to 1.3 copies/ll (1:10,000 dilution).
Applying these primers to the CSF of the 10 CFS samples from the 43 samples yielded negative results. A second set of primers designed for XMRV, but different
Volume 69, No. 4
Schutzer et al: XMRV in CSF in Chronic Fatigue
from the duplicated published sequences, also yielded
negative results for these 10 samples.
Blinded Independent Evaluation
of Samples for XMRV by PCR
Nucleic acid extracts from 43 CFS patients that were
pooled into 2 groups, positive controls (distilled water
and XMRV cDNA), and negative controls (distilled
water) were all negative except for the spiked control
sample, containing cDNA from XMRV.
Blinded Independent Evaluation of Extracts
for XMRV by PCR after Cocultivation
Cocultivation experiments also yielded negative results.
Discussion
Since the first reports by Lombardi et al,2 there have
been several other publications with varying results
related to the presence of XMRV or MRV in the peripheral blood cells or plasma in patients with CFS.3,13–17
Very recent studies are emerging that support detection
of viral sequences, but relate this to a laboratory contamination from mouse nucleic acid.4–6 However, no studies
have been published to date probing for the presence of
XMRV in the CSF. As we have already noted, finding
this retrovirus in the CSF compartment would be a first
step to determine if it is responsible for the pathogenesis
of the prominent symptoms, which appear to be CNS
related. Determining that the retrovirus is not present by
sensitive methods would suggest that exploration of other
causes or pathogenetic mechanisms is warranted. As we
also noted, should XMRV be detected in the CSF, a reasonable question toward determining potential pathogenesis would be whether XMRV is found alone or there
are other ubiquitous viruses present. The detection or absence of several other viruses is important in and of itself.
However, in the context of an XMRV study, the presence
of many other viruses would open the discussion of
opportunistic presence as opposed to a causative relation.
Using several techniques, we could not find any
evidence that XMRV was present in the CSF of a welldefined group of CFS patients. We also did not find evidence of any other common viruses in these samples.
This absence of detection was also supported by the laboratory that included members of the original discovery
team. We are aware that even presuming the validity of
the combined methods, XMRV could be present but
below the limits of detection. We are also aware that
CFS may be a heterogeneous condition, and that it is
possible that select populations may be found to have
XMRV or other viruses associated with it. Although we
did not detect the virus, and therefore contamination is
April 2011
not an issue here, we do have contingency methods in
place to remove potential contaminating microbial
nucleic acids that may be present even in commercial
reagents, such as Sato et al found in kits used for
XMRV.18 Currently, there are no published biological
markers to distinguish CFS, thereby contributing to the
difficulty in selecting populations for investigation as to
the presence of XMRV or other infectious agents. The
only other neurologic study to have been investigated is
amyotrophic lateral sclerosis,19 and no XMRV was
detected. These findings should be considered in the context of the known human retrovirus, human immunodeficiency virus, where CSF detection of the virus is relatively easy to accomplish. A recent study was still able to
detect nucleic acid of that virus in the CSF of individuals
even as they were undergoing therapy that decreased viral
burden, at least peripherally.20
In summary, our data are suggestive but not definitive evidence that pathogenesis of CFS, as relates to
the CNS, is not directly linked to XMRV, and other
potential options independent of a CNS infection with
XMRV such as inflammatory mechanisms should be
considered. It would be advantageous, for future pathogenesis studies, to include sampling from the relevant
organ system when it seems to be involved, such as the
CNS in this case. Other viruses can be investigated
with some of the broad methods and other developing
technologies.
Acknowledgment
This study was supported by the National Institute of
Allergy and Infectious Diseases, National Institutes of
Health (grants AI088765 and AI32247). We appreciate
VIP Dx providing an XMRV cDNA control and processing the samples provided to them in a blinded fashion.
Potential Conflicts of Interest
DE, ME, and MR are employees of the company, Ibis
Biosciences.
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