Journal of Medical Virology 9999:1–9 (2013)
Randomized Clinical Trial to Evaluate the Efficacy
and Safety of Valganciclovir in a Subset of Patients
With Chronic Fatigue Syndrome
Jose G. Montoya,1,2* Andreas M. Kogelnik,2Munveer Bhangoo,2Mitchell R. Lunn,2,3y
Louis Flamand,4Lindsey E. Merrihew,2,3Tessa Watt,2Jessica T. Kubo,1,3,5Jane Paik,1,3,5
and Manisha Desai1,3,5
1Department of Medicine, Stanford University School of Medicine, Stanford, California
2Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford,
3Stanford University School of Medicine, Stanford, California
4Department of Microbiology, Infectious Diseases and Immunology, Faculty of Medicine, Laval University, Que ´bec,
5Division of General Medicine Disciplines and Quantitative Sciences Unit, Stanford University School of Medicine,
There is no known
fatigue syndrome (CFS). Little is known about
its pathogenesis. Human herpesvirus 6 (HHV-6)
proposed as infectious triggers. Thirty CFS
against HHV-6 and EBV were randomized 2:1
to receive valganciclovir (VGCV) or placebo for
6 months in a double-blind, placebo-controlled
trial. Clinical endpoints aimed at measuring
physical and mental fatigue included the Multi-
dimensional Fatigue Inventory (MFI-20) and
Fatigue Severity Scale (FSS) scores, self-re-
ported cognitive function, and physician-deter-
mined responder status. Biological endpoints
included monocyte and neutrophil counts and
cytokine levels. VGCV patients experienced a
greater improvement by MFI-20 at 9 months
from baseline compared to placebo patients
but this difference was not statistically signifi-
cant. However, statistically significant differ-
ences in trajectories between groups were
observed in MFI-20 mental fatigue subscore
(P ¼ 0.039), FSS score (P ¼ 0.006), and cogni-
tive function (P ¼ 0.025). VGCV patients experi-
enced these improvements within the first
3 months and maintained that benefit over the
remaining 9 months. Patients in the VGCV arm
were 7.4 times more likely to be classified as
responders(P ¼ 0.029).
monocyte counts decreased (P < 0.001), neu-
trophil counts increased (P ¼ 0.037) and cyto-
kines were more likely to evolve towards a
Th1-profile (P < 0.001). Viral IgG antibody titers
did not differ between arms. VGCV may have
treatment for chronic
clinical benefit in a subset of CFS patients
independent of placebo effect, possibly medi-
ated by immunomodulation and/or antiviral
effect. Further investigation with longer treat-
ment duration and a larger sample size is
warranted. J. Med. Virol. 9999:1–9, 2013.
# 2013 Wiley Periodicals, Inc.
Additional supporting information may be found in the online
version of this article at the publisher’s website.
Grant sponsor: F. Hoffmann–La Roche (Basel, Switzerland);
Grant sponsor: Stanford University School of Medicine Molecular
Basis of Medicine Scholarly Concentration; Grant sponsor:
Stanford University School of Medicine Medical Scholars Fellow-
yHarvard Medical School, 25 Shattuck Street, Boston, MA
The present address of Tessa Watt is University of Michigan
Medical School, 1301 Catherine Road, Ann Arbor, MI, 48109
The present address of Mitchell R. Lunn is Department of
Medicine, Brigham and Women’s Hospital, 75 Francis Street,
Boston, MA, 02115
The present address of Munveer Bhangoo is University of
California-San Diego School of Medicine, 9500 Gilman Drive, La
Jolla, CA, 92093
The present address of Andreas M. Kogelnik is Open Medicine
Institute, 2500 Hospital Drive - Building 2, Mountain View, CA,
?Correspondence to: Jose G. Montoya, Department of Medicine,
Stanford University School of Medicine, Stanford, CA 94305.
Accepted 18 June 2013
Published online in Wiley Online Library
C 2013 WILEY PERIODICALS, INC.
valganciclovir; chronic fatigue
syndrome; human herpesvirus
6; Epstein–Barr virus; rando-
mized clinical trial
Chronic fatigue syndrome (CFS)—a chronic, complex,
and incapacitating illness of unknown etiology—is
characterized by profound physical fatigue that is not
improved by bed rest and can be significantly worsened
by physical or mental activity [Fukuda et al., 1994].
Little is known about its pathogenesis and diagnosis is
based on clinical symptoms as opposed to an objective
biomarker [Klimas et al., 2012]. Treatment regimens
are divergent, often deemed controversial, and usually
not supported by randomized, placebo-controlled clinical
trials [Straus et al., 1988].
Infection has been long suspected to be a trigger of
CFS, as many patients recall the onset of their
syndrome as an “influenza-like” illness, and out-
breaks of CFS have been reported in community and
hospital settings [Briggs and Levine, 1994; Levine,
1994; Kerr et al., 2002; Hickie et al., 2006; Komaroff,
2006; Katz et al., 2009; Komaroff and Cho, 2011]. An
aberrant immune response against periodic reactiva-
tion of or re-infection with an infectious agent(s) has
been proposed as a mechanism responsible for the
perpetuation and fluctuation of the CFS symptoms
[Tobi et al., 1982; Patnaik et al., 1995; Buchwald
et al., 1996; Komaroff and Cho, 2011]. Thus, long-
term and pathogen-directed interventions have been
attempted in subsets of CFS patients who meet
certain laboratory markers for a given organism with
the hope to significantly ameliorate or end suffering
[Kogelnik et al., 2006; Lerner et al., 2007].
It has been postulated that elevated IgG antibody
titers against human herpesvirus 6 (HHV-6) and EBV
could be interpreted as indicators of their periodic
reactivation or re-infection in CFS patients [Straus
et al., 1985; Buchwald et al., 1996] and that valganci-
clovir (VGCV) would suppress their reactivation or
treat re-infection [Lerner et al., 2002; Montoya, 2007].
In an open-label study, a significant clinical improve-
ment in this subset of CFS patients was observed
following 6 months of VGCV treatment [Kogelnik
et al., 2006]. However, it was not possible to conclude
whether an antiviral, immunomodulatory, or placebo
effect of the drug mediated this benefit. In this study,
the efficacy and safety of VGCV in this subset of CFS
patients when compared to placebo was examined and
biological endpoints that could identify potential
mechanisms of action were explored.
Study Protocol and Patients
The evaluation of valganciclovir in longstanding viral
exposure (EVOLVE) study was an investigator-initiated,
randomized, double-blind, placebo-controlled, clinical
trial to evaluate the efficacy and safety of VGCV in
CFS patients who have elevated IgG antibody titers
against HHV-6 and EBV. The Stanford University
Institutional Review Board (IRB) approved the study.
May 2007 and July 2007. Data collection was com-
pleted in April 2008.
Patients were eligible for inclusion if they (1) were
18 years of age or older, (2) met the CFS case
definition established in 1994 [Fukuda et al., 1994],
(3) had suspected viral onset (“influenza-like” or viral
illness diagnosed by a physician) of CFS, and (4) had
elevated antibody titers that fit one of the following
schema(i) HHV-6IgG ? 1:640,
? 1:640, and EBV EA IgG ? 1:160 or (ii) HHV-6
IgG ? 1:320, EBV VCA IgG ? 1:1,280 and EBV EA
IgG ? 1:160 (for further information see Supplemen-
tary Material Document 1). Figure 1 describes the
process for patient referral, screening, enrolment,
and allocation into the study. One hundred fifty-five
patients were referred to the study. Of the 155, 110
were excluded because the initial screening revealed
low antibody titers. Fifteen additional patients were
excluded because of low antibody titers on repeat
testing (five patients), exclusionary comorbidities (3),
conflicting medication (3), patients declined to partici-
pate (2), study was full (2; Fig. 1). The three patients
with exclusionary comorbidities had the diagnosis of:
uncontrolled hypothyroidism, uncontrolled major de-
pression, and hepatitis C.
Thirty patients were enrolled and randomized in a
2:1 manner to be treated with either VGCV (20
patients) or placebo (10 patients).
Patients were given VGCV or placebo based on
their assignment for 6 months and followed for 6
additional months. Patients and investigators were
blinded for a total of 9 months from the start of
randomization and until data were collected and
locked onto three CDs. The packaging of VCGV and
placebo was performed by Roche at their headquar-
ters (Basel, Switzerland) and sent to the Stanford
Pharmacy. VCGV or identical-appearing placebo was
initiated at a dose of 900 mg (two 450 mg tablets)
twice daily for 21 days followed by 900 mg once daily
to complete 6 months.
and enrolled between
In the absence of widely-accepted endpoints for
randomized clinical trials involving CFS patients, the
Multidimensional Fatigue Inventory (MFI-20, for
Document 2) score change at 9 months from baseline
was chosen as the primary outcome of interest
because investigators at the Centers for Disease
Control and Prevention (CDC) had validated previ-
ously the MFI-20 as an instrument that identifies
CFS in a reproducible manner [Gentile et al., 2003;
Reeves et al., 2005; Lin et al., 2009]. The MFI-20
J. Med. Virol. DOI 10.1002/jmv
2 Montoya et al.
score assesses general fatigue, physical fatigue, men-
tal fatigue, reduced motivation, and reduced activity.
Higher values indicate increased severity. The MFI-
20 mental fatigue subscore uses data already collect-
ed in the overall MFI-20 questionnaire but primarily
assesses cognitive, rather than physical fatigue. The
following additional (i.e., secondary) clinical measure-
ments were considered: CDC CFS Symptom Inventory
(CDC CFS SI), Fatigue Severity Scale (FSS, for
Document 3), Hamilton Rating Scale for Depression
(HAM-D), self-reported physical functioning and cog-
nitive functioning scores assessed by study personnel
during the study visit on a scale from 1% to 100%,
the Paced Auditory Serial Addition Test (PASAT) and
the Sleep Assessment Questionnaire (SAQ). These
additional endpoints were chosen because they have
been used in CFS studies. In this study, their use for
the first time in a randomized clinical trial is
reported. Clinical endpoints were collected at weeks
0, 1, 2, 3, 4, 8, 12, 24, and 48.
Finally, prior to unblinding, all subjects were
classified as a responder or non-responder by a
formal committee (comprised of four members of the
Referred to the study (n=155)
Assessed for eligibility (n=45)
Allocated to treatment (n=20)
• Received treatment (n=20)
Allocated to placebo (n=10)
• Received placebo (n=10)
Discontinued treatment (n=1)
• Developed ovarian cancer at
• None excluded in intention-
Discontinued placebo (n=1)
• Developed breast cancer at
• None excluded in intention-
• Low repeat antibody titers (n=5)
• Exclusionary comorbidities (n=3)
• Conflicting medication (n=3)
• Decline to participate (n=2)
• Study full (n=2)
• Low antibody titers (n=110)
evaluate the efficacy and safety of valganciclovir in patients with chronic fatigue syndrome and
elevated antibody titers against human herpes virus 6 (HHV-6) and Epstein–Barr virus (EBV).
Screening, enrolment, and allocation of patients in a randomized clinical trial to
J. Med. Virol. DOI 10.1002/jmv
Valganciclovir in Chronic Fatigue Syndrome3
study team) who reviewed the entire profile of clinical
symptoms as scored by the MFI-20 scores for each
subject before unblinding (physician-determined re-
monocyte and neutrophil counts were measured as
part of routine complete blood cell counts performed
in the Stanford Anatomic Pathology & Clinical Labo-
ratories and collected at weeks 0, 1, 2, 3, 4, 8, 12, 16,
20, 24, 25, 36, and 48.
Serum cytokine levels were obtained
using a 37-multiplex array on the Luminex 200 IS
system (Affymetrix, Santa Clara, CA) performed at
the Stanford Human Immune Monitoring Center and
following the manufacturer’s instructions. Serum
samples for the cytokine assay was collected at 0, 1,
2, 3, 4, 8, 12, 16, 20, 24, 36, and 48.
Viral IgG antibody titers.
against EBV and HHV-6 were measured by immuno-
fluorescence assay (IFA) at focus diagnostics (Cy-
press, CA). IgG antibody titers against HHV-6 were
also analyzed by IFA at the laboratory of one of the
authors (L.F.). Serum to measure viral IgG antibody
titers was collected at weeks 0 and 24.
IgG antibodies titers
Adherence to Study Protocol, and Safety
Adherence was reinforced at each visit by direct
questioning and pill counts. Additionally, serum
samples were obtained from all subjects to measure
ganciclovir levels at weeks 3, 12, and 24 (VGCV is
the pro-drug of ganciclovir). To assess the safety of
the drug, complete blood cell counts with leukocyte
differential, renal function tests, and liver function
tests were performed per protocol. In addition, safety
issues were assessed at each visit.
Differences in baseline and clinical patient charac-
teristics between the VCGV and placebo arms were
evaluated by the Fisher’s exact test or a Student’s t-
test as appropriate. As many secondary outcomes
were considered corresponding to many hypotheses
tested, secondary analyses from this study serve a
hypothesis-generating purpose. As such, analyses are
considered exploratory, and P-values are descriptive.
All tests were two-sided and performed at the 0.05
level of significance.
To address the primary hypothesis that VGCV
clinically improves physical and mental fatigue in a
subset of CFS patients, an intent-to-treat analysis
and two statistical methods were used. Analysis of
variance (ANOVA) methods were used to determine
whether improvement in the VGCV arm occurred at
9 months in the clinical and biological endpoints.
Mixed-effects linear (MEL) regression methods were
used to address whether trajectories of these end-
points measured over time differed by treatment
group status and included a subject-specific random
intercept term to account for the correlation of
measurements within a subject over time. Two
models of this type were considered: the first as-
sumed a linear relation between the outcome and
time, and the second categorized time into four
clinically meaningful time periods—baseline, induc-
tion period (first 3 weeks on 900 mg twice daily),
maintenance period (last 21 weeks on 900 mg daily),
and post-treatment follow-up—and allowed the out-
come behavior to be nonlinear over time when
Demographics and Clinical Parameters of the
Patients at Baseline
Demographic and baseline clinical characteristics
by treatment assignment are shown in Table I.
were not statistically different between the VGCV
and placebo arms with the exception of smoking
history. The VGCV arm experienced a higher propor-
tion of individuals with history of smoking but only
three were current smokers; no one in the placebo
arm was a current or former smoker.
Adherence of Subjects to Treatment Assignment
In the VGCV arm, all of the subjects had detectable
ganciclovir levels after 3 weeks of treatment (mean
¼ 5.08 mg/ml, SD ¼ 3.47), 18 had detectable levels at
week 12 (mean ¼ 2.34 mg/ml, SD ¼ 2.46), and 13 at
week 24 (mean ¼ 1.10 mg/ml, SD ¼ 1.62). Unexpect-
edly, three subjects in the placebo arm had low but
detectable levels ranging from 0.2 to 0.3 mg/ml; the
remaining seven had undetectable levels (<0.1 mg/ml)
at all time points.
In Tables II and III, relevant changes in clinical
outcomes for the VGCV and placebo arms are
reported. The MFI-20 total score (primary outcome)
revealed a greater improvement in the VGCV arm
(?6.15 [SD 12.06]) relative to the placebo arm (?1.10
[SD 5.90]) at 9 months compared to baseline, howev-
er, this difference in improvement was not statisti-
cally significant (Table II) by ANOVA or MEL
regression model (P ¼ 0.114; Fig. 2A). Statistically
significant differences between arms were observed
in the trajectories of MFI-20 mental fatigue subscore,
(Fig. 2B–D) in the MEL regression model.
improvements in the VGCV arm compared to the
placebo arm (P ¼ 0.039) despite different but not
statistically significant baselines (Fig. 2B; for the
J. Med. Virol. DOI 10.1002/jmv
4 Montoya et al.
MFI-20 mental fatigue subscore of all patients,
see Supplementary Material Fig. 1). The benefit
in the VGCV arm was observed within the first
3 months post-randomization. The VGCV arm had
slightly larger decreases in fatigue as measured
by the FSS score than the placebo arm (P ¼ 0.006;
Fig. 2C). In the MFI-20 mental fatigue subscore
and FSS scores, lower values indicate decreased
severity. The VGCV arm also experienced greater
increases in self-reported cognitive functioning score
over time compared to the placebo arm (1.72 points
vs. 0.59 per month, respectively; P ¼ 0.025; Fig. 2D).
VGCV patients were 7.4 times more likely to be
(P ¼ 0.029).
Of interest, patients in the VGCV and placebo
arms appear to have experienced initial worsening of
their CFS symptoms within the first 2 months of the
initiation of the trial (Fig. 2A–D). This initial worsen-
ing was followedbya
(reflected in the MFI-20 mental fatigue subscore, FSS
score, and self-reported cognitive function) in patients
in the VGCV arm but was not observed in patients in
the placebo arm (Fig. 2B–D).
clinical scores including CDC CFS SI, HAM-D score,
mean self-reported physical functioning assessment,
PASAT, or SAQ, the VGCV arm did not have a
greater improvement than the placebo arm.
Monocyte and neutrophil counts.
the VGCV arm experienced a statistically significant
followed by a transient increase during the post-
absolute neutrophil counts (P ¼ 0.037; Fig. 3 A,B).
counts(P < 0.001)
TABLE II. Primary Outcome and Physician-Determined
Responder Status Prior to Unblinding
change in MFI-20 total
score at 9 monthsa
Likelihood of being
classified as a
responder by the study
status prior to
aStatistical significance was assessed by analysis of variance
TABLE I. Demographics and Clinical Parameters at
Sample size, n
Number lost to
History of smoking
Mean age at study
Mean age at viral
Mean duration of
Mean BMI, kg/m2
MFI-20 Total Score
37.48 (9.12)34.94 (10.74) 0.503
12.70 (10.02) 13.53 (7.82)0.820
?P-values correspond to either Fisher’s exact test or a t-test as
TABLE III. Differences in Clinical Outcomes Over Time in
the Valganciclovir and Placebo Arms Prior to Unblinding
Effect over time
by study arm
Valganciclovir Placebo P-value†
Fatigue severity scale
CDC symptom inventory scores
Sleep assessment ques-
Hamilton Rating Scale for Depression
PASAT, Paced Auditory Serial Addition Test; HAM-D, Hamilton
Rating Scale for Depression.
†P-value for interaction effect indicating whether changes in
outcome over time vary by study arm. Statistical significance was
assessed by mixed-effects linear regression model (MEL).
J. Med. Virol. DOI 10.1002/jmv
Valganciclovir in Chronic Fatigue Syndrome5
comparable between arms with the exception of two.
Baseline cytokine levels for tumor necrosis factor
(TNF)-a and interleukin (IL)-17F were higher in the
VGCV arm than the placebo arm by factors of 1.30
and 1.42, respectively (P ¼ 0.03 and P ¼ 0.04, respec-
tively). This study interest, however, was whether
changes in cytokine levels over the 9-month period
differed between the arms. Cytokines that significant-
ly varied over time by treatment status (P < 0.05)
are shown in (see Supplementary Table I). Among
the 37 cytokines, two important families or groupings
of cytokines have been previously identified as rele-
vant in other studies of immune function in CFS
patients [Broderick et al., 2010; Brenu et al., 2011].
Therefore, the impact of VGCV on trajectories of the
Th1 (IL-2, IL-12, IFN-g)- and Th2 (IL-4, IL-5, IL-6,
IL-10, IL-13)-associated cytokines over time was
assessed. Levels for each “family” (Th1-associated vs.
Th2-associated) were derived by summing the levels
of the cytokines within each “family.” Th1 and Th2
were not significantly different between the treat-
ment arms at baseline (P ¼ 0.361 and P ¼ 0.127,
respectively). A 2.52-fold increase over a 9-month
period in Th1-type cytokines in the VGCV arm and a
1.48-fold decrease in the placebo arm (P < 0.001) was
Baseline cytokine levels were largely
found. No significant difference was observed for Th-
2 type cytokines.
Viral IgG antibody titers.
body levels between baseline and 6 months measure-
ments (ANOVA) and in their trajectories (MEL
regression model) over a 6-month period were not
Differences for anti-
VGCV was well-tolerated and was not discontinued
due to hematologic or hepatic adverse events. Two
patients were diagnosed with cancer during the
study period: in the VGCV arm, one patient was
removed from the study at week 16 due to the
diagnosis of ovarian cancer; in the placebo arm, one
patient was diagnosed with breast cancer at week 36.
These two serious adverse events were deemed
unrelated to VGCV.
Antiviral therapy for patients with CFS is widely
viewed as unnecessary and is unsupported by clinical
trials with rigorous study designs. Straus et al.
 reported that acyclovir lacked efficacy for the
treatment and placebo (control) arms. B: Mean cognitive
function in the valganciclovir treatment arm compared to
the placebo (control) arm as measured by the Multidimensional
Fatigue Inventory (MFI-20) mental fatigue subscore (points).
Higher valuesin the MFI-20
indicate increased severity. C: Mean physical fatigue in the
A: Mean MFI-20 total score in the valganciclovir
valganciclovir treatment arm compared to the placebo (control)
arm. Physical fatigue was measured by the Fatigue Severity
Scale (FSS) score. Higher values in the FSS score indicate
increased severity. D: Mean self-reported cognitive functioning
average score in the valganciclovir treatment and placebo
J. Med. Virol. DOI 10.1002/jmv
6 Montoya et al.
treatment of CFS patients in a randomized placebo-
controlled trial. However, acyclovir administration
duration was only 5 weeks, and the possibility of
HHV-6 infection (a virus known to be essentially
unaffected in vitro by acyclovir) was not investigated.
Since then, several investigators have reported poten-
tial benefit of various antivirals used for longer
periods in open-label observations [Kogelnik et al.,
2006; Lerner et al., 2010].
Findings in this study suggest that VGCV may
have a clinical benefit, independent from placebo in
that subset of patients with CFS who have serological
evidence of reactivated EBV and/or HHV-6 infection.
With regard to the physical (i.e., FSS score) and
mental fatigue (i.e., MFI-20 mental fatigue subscore,
self-reported cognitive function) outcomes, these find-
ings consistently indicated larger improvements in
the VGCV arm. Most notable was the difference in
proportion of responders between the arms: all but 2
of the 15 responders were in the VGCV arm.
While differences between arms in the originally
chosen primary endpoint (i.e., change in MFI-20 at
month 9 compared to baseline assessed by ANOVA)
were not found, compelling differences were found in
the trajectories of MFI-20 and FSS scores by MEL
regression analysis. While these findings may seem
contradictory, they are not. The primary endpoint
investigated the effect of treatment on change in
outcomes at a specific time point (9 months), whereas
MEL regression analysis evaluated the effect of
treatment on an entire trajectory of the particular
outcome over time. CFS is characterized by a highly
fluctuating clinical course; symptoms vary significant-
ly on a daily, weekly, and monthly basis. Therefore,
MEL regression models that make use of all data
points appear to be more suitable to determine drug
effect over placebo in CFS patients.
Patients in the VGCV and placebo arms experi-
enced an initial worsening of their symptoms that
has been previously reported by the Stanford CFS
group and in a recent clinical trial [Kogelnik et al.,
2006; Fluge et al., 2011]. It is possible that the
worsening in the placebo arm was due to placebo
effect and/or the additional physical/emotional load
of frequent visits to the clinical research center. In
the VGCV arm, in addition to the factors present in
the placebo group, a drug effect may have taken
place as well. The pathogenesis of this initial worsen-
ing is unclear but it may resemble a Jarisch–
Herxheimer-like reaction that has been observed
during the initial treatment of certain infections and
may be mediated by an immune response to tran-
siently increased circulating
In addition, these results suggest possible mecha-
nisms for the clinical benefit observed in the VGCV
arm. Monocytopenia, neutrophilia, and differences in
Th1-related cytokines over time were associated with
the use of VGCV. In immunocompromised patients,
ganciclovir (the active drug in VGCV) is a commonly
used antiviral against herpesviruses and appears to
work by interfering with viral DNA chain elongation
[Montoya, 2007; Razonable, 2011]. In immunocompro-
mised patients, ganciclovir frequently contributes to
leukopenia and neutropenia and it is not known to
cause monocytopenia or neutrophilia. Monocytes are
known to be targeted by HHV-6 [Kondo et al., 2002;
Janelle and Flamand, 2006] and can be infected by
EBV [Savard et al., 2000; Tugizov et al., 2007; Wall-
ing et al., 2007]. Thus, it is possible that in CFS
patients HHV-6 and EBV are circulating in peripher-
al blood within monocytes. Since monocytes are
transformed into macrophages in tissues, including
the central nervous system, by lowering monocytes in
peripheral blood, VGCV may be indirectly decreasing
the viral HHV-6/EBV burden in the tissues of CFS
patients. In addition, by decreasing influx of infected
monocytes (with the capacity of triggering inflamma-
tion) into affected tissues, VGCV may be contributing
towards the restoration of a more effective and
healthier local immune response.” The neutrophilic
response observed in the VGCV arm was unexpected
but was also validated by the increase of ENA-78, a
known neutrophil chemoattractant [Liu et al., 2009].
The antiviral role of neutrophils is becoming increas-
ingly appreciated [Butler et al., 2011] and should not
come as a surprise given the tendency of several
viruses to cause leukopenia. The trend towards a Th1
cytokine profile in the VGCV arm would reverse the
Monocyte count (k/uL)
Neutrophil count (k/uL)
treatment and placebo (control) arms. B: Mean neutrophil count
in the valganciclovir treatment and control arms.
A: Mean monocyte count in the valganciclovir
J. Med. Virol. DOI 10.1002/jmv
Valganciclovir in Chronic Fatigue Syndrome7
Broderick et al. in CFS patients [Broderick et al.,
2010; Brenu et al., 2011]. Significant declines in the
HHV-6 or EBV antibody titers at 6 months were not
observed, suggesting that their decline requires lon-
ger periods of VGCV administration and/or that
VGCV primarily works through immunomodulatory
properties in CFS patients. Alternatively, it is possi-
ble that HHV-6 and EBV antigens were circulating
forming immune complexes resulting in artificially
depressed levels of antibodies; thus, when VGCV
reduced load of circulating Ag, antibody levels may
have risen because less antibodies were bound in the
A study by Fluge et al.  suggested that use of
rituximab was associated with significant clinical
benefit. Monocytopenia in this study and depletion of
B cells in theirs, would suggest that excesses or
abnormalities in antigen presentation might be a key
underlying mechanism in CFS.
This study has several limitations including the
small sample size and testing of numerous explorato-
ry hypotheses. However, the randomized, double-
blind, placebo-controlled, study design permits that
these findings be worthy of further exploration. A
limitation, against a stronger VGCV benefit, was that
three patients in the placebo arm had detectable
ganciclovir levels. Upon thorough and careful ques-
tioning, these patients reported no usage of VGCV or
ganciclovir outside the study setting.
Findings in this study suggest that clinical trials
using longer courses of VGCV and a larger sample
size are warranted. They also suggest that outcomes
be analyzed by MEL regression models (or similar
methods) and that MFI-20 scores/subscores and the
FSS score be used among clinical endpoints. Results
in this study also support the view that CFS is a real
disease that necessitates sound translational research
and that can be amenable to medical interventions.
Hoffmann-La Roche (Basel, Switzerland) for unre-
stricted financial support to perform the study and
providing the valganciclovir and placebo drugs; Kris-
tin Loomis and Dharam Ablashi, Ph.D. at the HHV-6
Foundation for facilitating the initial meeting with
Roche and their scientific and logistical support of
several years; Martha Hamilton, Pharm.D. at the
Stanford Hospital and Clinics Pharmacy Services for
assuring the study drug was delivered on time to the
Maecker, Ph.D., and Yael Rosenberg-Hasson at the
Stanford Institute for Immunity, Transplantation
and Infection and Stanford Human Immune Monitor-
ing Center (HIMC) for the integrity of the cytokine
data and inspiration to continue our endeavors
against existing dogmas in the field of CFS. Special
thanks to CFS patients who have been patiently
waiting for too long for sound answers from medical
and scientific communities; Michaela Kiernan, Ph.D.
at the Stanford Prevention Research Center for her
critical review of the entire manuscript and superb
comments that made our manuscript much more
clear and reader friendly; and Geronimo Terres,
Ph.D. for his thoughtful comments on the kinetics of
the viral antibody titers. M.R.L. was supported by
the Stanford University School of Medicine Molecular
Basis of Medicine Scholarly Concentration and the
Scholars Fellowship Program.
Registration, investigational new drug application,
and role of F. Hoffmann–La Roche Ltd: EVOLVE was
#NCT00478465. Authorization to use VGCV was
provided under the United States Food and Drug
Administration Investigational New Drug Application
#77,331 (J.G.M.). F. Hoffmann–La Roche (Roche;
Basel, Switzerland) provided financial support for the
study as well as VGCV and its matching placebo,
approved the study design proposed by the investiga-
tors, and performed randomization. Stanford investi-
gators and authors independently executed the study;
collected, locked, and analyzed the data; and wrote
the manuscript. Adverse events were reported to and
investigated by the Stanford IRB and Roche within
24 hr. Prior to unblinding, data were locked and
recorded onto three CDs, which are currently kept at
Stanford (2 CDs) and Roche (1 CD). The study was
conducted in full conformance with the “Declaration
of Helsinki,” the “Guidelines for Good Clinical Prac-
tice” harmonized Tripartite Guideline (1997), and
Brenu EW, van Driel ML, Staines DR, Ashton KJ, Ramos SB,
Keane J, Klimas NG, Marshall-Gradisnik SM. 2011. Immunolog-
ical abnormalities as potential biomarkers in chronic fatigue
syndrome/myalgic encephalomyelitis. J Transl Med 9:81–90.
Briggs NC, Levine PH. 1994. A comparative review of systemic and
neurological symptomatology in 12 outbreaks collectively de-
scribed as chronic fatigue syndrome, epidemic neuromyasthenia,
and myalgic encephalomyelitis. Clin Infect Dis 18:S32–S42.
Broderick G, Fuite J, Kreitz A, Vernon SD, Klimas N, Fletcher MA.
2010. A formal analysis of cytokine networks in chronic fatigue
syndrome. Brain Behav Immun 24:1209–1217.
Bryceson AD. 1976. Clinical pathology of the Jarisch–Herxheimer
reaction. J Infect Dis 133:696–704.
Buchwald D, Ashley RL, Pearlman T, Kith P, Komaroff AL. 1996.
Viral serologies in patients with chronic fatigue and chronic
fatigue syndrome. J Med Virol 50:25–30.
Butler LM, Jeffery HC, Wheat RL, Rae PC, Townsend K, Alkharsah
KR, Schulz TF, Nash GB, Blackbourn DJ. 2011. Kaposi’s
sarcoma-associated herpesvirus infection of endothelial cells
inhibits neutrophil recruitment through an interleukin-6-depen-
dent mechanism: A new paradigm for viral immune evasion. J
Fluge Ø, Bruland O, Risa K, Storstein A, Kristoffersen EK,
Storstein A, Kristoffersen EK, Sapkota D, Næss H, Dahl O,
Nyland H, Mella O. 2011. Benefit from B-lymphocyte depletion
using the anti-CD20 antibody rituximab in chronic fatigue
syndrome. A double-blind and placebo-controlled study. PLoS
Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff
A. 1994. The chronic fatigue syndrome: A comprehensive
approach to its definition and study. International Chronic
Fatigue Syndrome Study Group. Ann Intern Med 121:953–959.
J. Med. Virol. DOI 10.1002/jmv
8 Montoya et al.
Gentile S, Delaroziere JC, Favre F, Sambuc R, San Marco JL. 2003.
Validation of the French ’multidimensional fatigue inventory’
(MFI 20). Eur J Cancer Care (Engl) 12:58–64.
Hickie I, Davenport T, Wakefield D, Vollmer-Conna U, Cameron B,
Vernon SD, Reeves WC, Lloyd A. 2006. Post-infective and
chronic fatigue syndromes precipitated by viral and non-viral
pathogens: Prospective cohort study. BMJ 333:575–581.
Janelle ME, Flamand L. 2006. Phenotypic alterations and survival
of monocytes following infection by human herpesvirus-6. Arch
Katz BZ, Shiraishi Y, Mears CJ, Binns HJ, Taylor R. 2009. Chronic
fatigue syndrome after infectious mononucleosis in adolescents.
Kerr JR, Bracewell J, Laing I, Mattey DL, Bernstein RM, Bruce IN,
Tyrrell DA. 2002. Chronic fatigue syndrome and arthralgia
following parvovirus B19 infection. J Rheumatol 29:595–602.
Klimas NG, Broderick G, Fletcher MA. 2012. Biomarkers for
chronic fatigue. Brain Behav Immun 26:1202–1210.
Kogelnik AM, Loomis K, Hoegh-Petersen M, Rosso F, Hischier C,
Montoya JG. 2006. Use of valganciclovir in patients with
elevated antibody titers against Human Herpesvirus-6 (HHV-6)
and Epstein–Barr Virus (EBV) who were experiencing central
nervous system dysfunction including long-standing fatigue. J
Clin Virol 37:S33–S38.
Komaroff AL. 2006. Is human herpesvirus-6 a trigger for chronic
fatigue syndrome? J Clin Virol 37:S39–S46.
Komaroff AL, Cho TA. 2011. Role of infection and neurologic
dysfunction in chronic fatigue syndrome. Semin Neurol 31:325–
Kondo K, Kondo T, Shimada K, Amo K, Miyagawa H, Yamanishi K.
2002. Strong interaction between human herpesvirus 6 and
peripheral blood monocytes/macrophages during acute infection.
J Med Virol 67:364–369.
Lerner AM, Beqaj SH, Deeter RG, Dworkin HJ, Zervos M, Chang
CH, Fitzgerald JT, Goldstein J, O’Neill W. 2002. A six-month
trial of valacyclovir in the Epstein–Barr virus subset of chronic
fatigue syndrome: Improvement in left ventricular function.
Drugs Today 38:549–561.
Lerner AM, Beqaj SH, Deeter RG, Fitzgerald JT. 2007. Valacyclovir
treatment in Epstein–Barr virus subset chronic fatigue syn-
drome: Thirty-six months follow-up. In Vivo 21:707–713.
Lerner AM, Beqaj SH, Fitzgerald JT, Gill K, Gill C, Edington J.
2010. Subset-directed antiviral treatment of 142 herpesvirus
patients with chronic fatigue syndrome. Virus Adapt Treat 2:47–
Levine PH. 1994. Summary and perspective: Epidemiology of
chronic fatigue syndrome. Clin Infect Dis 18:S57–S60.
Lin JM, Brimmer DJ, Maloney EM, Nyarko E, Belue R, Reeves
WC. 2009. Further validation of the multidimensional fatigue
inventory in a US adult population sample. Popul Health Metr
Liu GN, Shi HZ, Xie ZH, Shen HH, Huang HQ, Deng JM, Liang QL,
Wu YB. 2009. Epithelial neutrophil-activating peptide-78 recruits
neutrophils into pleural effusion. Eur Respir J 34:184–190.
Montoya JG. 2007. Improving the tools in the fight against
cytomegalovirus or strengthening David to defeat Goliath. Curr
Opin Infect Dis 20:397–398.
Patnaik M, Komaroff AL, Conley E, Ojo-Amaize EA, Peter JB.
1995. Prevalence of IgM antibodies to human herpesvirus 6
early antigen (p41/38) in patients with chronic fatigue syn-
drome. J Infect Dis 172:1364–1367.
Razonable RR. 2011. Antiviral drugs for viruses other than human
immunodeficiency virus. Mayo Clin Proc 86:1009–1026.
Reeves WC, Wagner D, Nisenbaum R, Jones JF, Gurbaxani B,
Solomon L, Papanicolaou DA, Unger ER, Vernon SD, Heim C.
2005. Chronic fatique syndrome—A clinically emprical approach
to its definition and study. BMC Med 3:19–28.
Savard M, Belanger C, Tardif M, Gourde P, Flamand L, Gosselin J.
2000. Infection of primary human monocytes by Epstein–Barr
virus. J Virol 74:2612–2619.
Straus SE, Tosato G, Armstrong G, Lawley T, Preble OT, Henle W,
Davey R, Pearson G, Epstein J, Brus I, Blaese RM. 1985.
Persisting illness and fatigue in adults with evidence of
Epstein–Barr virus infection. Ann Intern Med 102:7–16.
Straus SE, Dale JK, Tobi M, Lawley T, Preble O, Blaese RM,
Hallahan C, Henle W. 1988. Acyclovir treatment of the chronic
fatigue syndrome. Lack of efficacy in a placebo-controlled trial.
New Engl J Med 319:1692–1698.
Tobi M, Morag A, Ravid Z, Chowers I, Feldman-Weiss V, Michaeli
Y, Ben-Chetrit E, Shalit M, Knobler H. 1982. Prolonged atypical
illness associated with serological evidence of persistent Ep-
stein–Barr virus infection. Lancet 1:61–64.
Tugizov S, Herrera R, Veluppillai P, Greenspan J, Greenspan D,
Palefsky JM. 2007. Epstein–Barr virus (EBV)-infected mono-
cytes facilitate dissemination of EBV within the oral mucosal
epithelium. J Virol 81:5484–5496.
Walling DM, Ray AJ, Nichols JE, Flaitz CM, Nichols CM. 2007.
Epstein–Barr virus infection of Langerhans cell precursors as a
mechanism of oral epithelial entry, persistence, and reactivation.
J Virol 81:7249–7268.
J. Med. Virol. DOI 10.1002/jmv
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