Illness Characterized by Fatigue, Neurologic and Immunologic Disorders,
and Active Human Herpesvirus Type 6 Infection
MD; Paul R. Cheney,
MD, PhD; Daniel L. Peterson, MD;
Berch Henry, PhD; Susan B.
Wormsley, BS; Ann Geiger,
BA; Dharam V. Ablashi. DVM;
S. Zaki Salahuddin, MS; Carl
Saxinger, PhD; Royce Biddle,
MD; Ron Kikinis, MD; Ferenc
A. Jolesz, MD; Thomas Folks,
PhD; N. Balachandran, PhD; James B. Peter, MD, PhD;
Robert C. Gallo, MD; and Anthony
L. Komaroff, MD
of Internal Medicine
15 January 1992; Vol 116 (2):103-13
Objective: To conduct neurologic, immunologic, and virologic studies
in patients with a chronic debilitating illness of acute onset.
Design: Cohort study with comparison to matched, healthy control
· Patients: We studied
259 patients who sought care in one medical practice; 29% of the
patients were regularly bedridden or shut-in.
Main Outcome Measures: Detailed medical history, physical
examination, conventional hematologic and chemistry testing, magnetic
resonance imaging (MRI) studies, lymphocyte phenotyping studies, and
assays for active infection of patients' lymphocytes with human
herpesvirus type 6 (HHV-6).
Main Results: Patients had a higher mean (+/- SD) CD4/CD8 T-cell
ratio than matched healthy controls (3.16 +/- 1.5 compared with 2.3 +/-
1.0, respectively; P < 0.003). Magnetic resonance scans of the brain
showed punctate, subcortical areas of high signal intensity consistent
with edema or demyelination in 78% of patients (95% Cl, 72% to 86%) and
in 21% of controls (Cl, 11% to 36%) (P < 10-9). Primary cell
culture of lymphocytes showed active replication of HHV-6 in 79 of 113
patients (70%; CI, 61% to 78%) and in 8 of 40 controls (20%; Cl, 9% to
36%) (P < 10-8), a finding confirmed by assays using
monoclonal antibodies specific for HHV-6 proteins and by polymerase
chain reaction assays specific for HHV-6 DNA.
Neurologic symptoms, MRI findings, and lymphocyte phenotyping studies
suggest that the patients may have been experiencing a chronic,
immunologically mediated inflammatory process of the central nervous
system. The active replication of HHV-6 most likely represents
reactivation of latent infection, perhaps due to immunologic
dysfunction. Our study did not directly address whether HHV-6, a
lymphotropic and gliotropic virus, plays a role in producing the
symptoms or the immunologic and neurologic dysfunction seen in this
illness. Whether the findings in our patients, who came from a
relatively small geographic area, will be generalizable to other
patients with a similar syndrome remains to be seen.
Annals of Internal Medicine. 1992:
For current author affiliations and addresses, see end
We studied 259
patients who had an illness that was typically of abrupt onset,
beginning with a “flu-like” syndrome that was followed by months or
years of sometimes disabling chronic fatigue and impaired cognition.
Enough cases occurred among family members, coworkers, and other close
contacts to suggest the possibility of an infectious agent transmissible
by casual contact. A few patients developed transient periods of
apparent encephalitis, characterized by confusion, ataxia, paresis, and
primary seizure disorders. Several unusual features have been revealed
by immunologic testing, magnetic resonance imaging (MRI) studies of the
brain, and virologic studies.
Initiation of the
In late 1984,
two of us (DLP and PRC) who had a general medicine practice in Incline
Village. Nevada, on the north shore of Lake Tahoe, began to see several
patients with an unusual illness. By mid-1985. we were concerned that an
epidemic might be unfolding. Two of us who were studying a similar
illness in an academic general medicine practice in Boston (DB and ALK)
heard of the events in Nevada. Together, the four of us began a formal
study in January 1986. A team from Boston traveled to Nevada on several
occasions. Many additional collaborators became involved. Research
assistants in Lake Tahoe and Boston gathered detailed clinical and
laboratory data and collected blood specimens, and all data were entered
into a computerized database in Boston. Because a formal study was
initiated a year after events unfolded, we were unfortunately not able
to determine exactly how many patients were seen in this practice who
had the symptoms of this unusual illness but were not entered into the
study. However, we estimate that 85% of the patients seen in the
practice who had this illness were enrolled.
One hundred and
eighty-three patients lived in one of several communities near the
California-Nevada border, in the vicinity of Lake Tahoe, and sought
medical care between 1984 and 1987 for chronic fatigue at the internal
medicine practice. The mean (+/- SD) age of this “Tahoe” group was 38.9
+/- 12.3 years: 67% were female and 41% were college graduates.
other patients with chronic, debilitating fatigue came to the same
practice during the same time period. These patients were from outside
the Lake Tahoe area, typically from urban areas of California and
Nevada. The mean age of this “non-Tahoe” group was 38.6 +/- 11.4 years:
70% were female and 36% were college graduates. The mean age of all
patients. including both the Tahoe and non-Tahoe groups, was 38.8 +/-
12.0 years: 68% of patients were female, and 39% were college graduates.
The Tahoe group
may have represented part of an epidemic, because most of these patients
became ill within a 2-year period and many had close contacts who became
ill. Hence, they are also referred to as the “epidemic” group; however,
the design of our study did not allow us to establish that a true
epidemic had occurred. The non-Tahoe group is referred to as the
“endemic” group because the patients did not know many other persons who
were similarly affected: however, many patients knew at least one close
contact who was allegedly affected.
patients enrolled in the study were asked to complete a detailed
questionnaire. and data from each patient's medical record were
abstracted. To be included in our study, a patient had to have chronic,
debilitating fatigue of at least 3 months duration that was associated
with at least two of the following symptoms (also of at least 3 months
duration): fever, headache, sore throat, earache. rhinorrhea, cough,
diarrhea, or myalgias. At the time of enrollment in the study, the
median duration of illness had already reached 1.3 years.
obtained from several different groups of healthy control subjects.
These control groups are described in the following sections and in
Table 1.with reference to the diagnostic testing for which they served
Table 1. Characteristics of Patients and
Control Subjects by Individual Test*
Patients Control P Value †
Mean age. y 39.5 +/-
11.2 37.5 +/- 10.7 > 0.2
70 53 0.07
Mean age, y 38.4 +/-
12.3 47.5 +/- 11.9 < 0.01
63 48 0.23
Mean age. y 41.2 +/-
11.7 32.4 +/- 9.1 ‡ < 0.01
70 60 ‡ 0.21
Mean age. y 38.8 +/-
10.8 36.9 +/- 13.2 > 0.2
72 68 > 0.2
HHV-6 = human herpesvirus-6: MRI = magnetic resonance imaging. Mean
values are expressed +/- SD.
P < 0.05 was considered statistically significant.
Data on age and sex were available from only 35 of the 54 control
Standard Laboratory Tests
Complete blood counts,
erythrocyte sedimentation rates, standard serum chemistry tests, various
tests for collagen vascular diseases. and thyroid function tests were
ordered when indicated clinically and not according to a study protocol.
Cerebrospinal Fluid Examination
Analysis of cerebrospinal
fluid obtained by lumbar puncture was done in six patients who had
developed acute neurologic changes (confusion, marked cognitive
deficits, delirium, ataxia, transient paresis, or primary seizure).
Magnetic Resonance Imaging of the Brain
imaging studies were done by one of us (RB) at Reno Diagnostics Center.
One hundred and forty-four patients had at least one MRI study. These
patients had somewhat greater debility but were otherwise generally
similar to the 1l5 patients in whom MRI studies were not done (Table 2).
The MRI findings in patients were compared with those in 47 control
subjects who did not have chronic fatigue and who were similar to the
patients in mean age and gender distribution (see Table 1). Forty-two of
these controls had been asked to undergo scanning as part of a study to
assess the resolution of a new machine with a larger magnet; four
patients had sustained head trauma and one patient had a suspected
cervical herniated disk. The images from the controls were obtained on
the same brand of machine, with the same size magnet, by Dr. Victor
Haughton of Milwaukee, Wisconsin. The images from patients could be
distinguished from those of control subjects by the logos of the two
radiology departments (Reno and Milwaukee); thus, blind interpretation
was not possible. However, the images were interpreted independently by
two of us (RB and FAJ), and inter-rater reliability was determined.
The patient studies were
done using a 1.5-tesla superconducting Signa magnetic resonance imager
(General Electric Medical, Milwaukee, Wisconsin). Images included a T1-
weighted sagittal scan followed by T2-weighted transaxial spin echo
scans of the brain. These axial scans were obtained at 5-mm intervals
from the foramen magnum to the vertex, with a skip of l to 2 mm. The
repetition time was between 2000 and 2500 ms and the echo delay time was
between 20 and 30 ms for the first echo and between 50 and 80 ms for the
Two of us (SBW and JBP)
did the phenotyping studies. Two hundred and thirty-nine measurements
were done in 121 patients who were slightly older but were otherwise
generally similar to the l39 patients not tested (Table 2). Studies were
also done on 270 occasions in 54 healthy control subjects (laboratory
personnel) who were somewhat younger but similar in gender distribution
to the patients (see Table 1). Phenotyping of coded specimens was done
concurrently and in blinded fashion by the same technicians using the
same reagents and flow cytometer.
Mononuclear cells were
separated by Ficoll-Hypaque centrifugation from 20 to 40 ml. of
heparinized venous blood and were then washed and stained with the
T-cell monoclonal antibodies CD2, CD4, CD8 (Ortho Pharmaceuticals,
Raritan, New Jersey), and CD5 (Boehringer Mannheim, Chicago, Illinois)
and the B-cell monoclonal antibody CD20 (Coulter Immunology, Hialeah,
Florida), using standard indirect immunofluorescence techniques,
isotype-matched myeloma protein was used in place of these monoclonal
antibodies as a staining control: fluorescein isothiocyanate-conjugated
goat anti-mouse immunoglobulin (Tago, Inc., Burlingame, California) was
the secondary reagent.
Assay for Active Replication of Human Herpesvirus Type 6
An assay was conducted by
one of us (BH) to detect active replication of HHV-6 in peripheral
mononuclear cells. Assays were done on at least one occasion in l13
patients. These patients were similar to the l46 patients not tested
(see Table 2). The only criterion in selecting patients for these
studies was the availability of the virology technician to receive and
process the blood sample immediately.
Heparinized blood samples
were obtained from 40 healthy blood donors and laboratory personnel who
served as controls and were similar to the patients in mean age and in
gender distribution (see Table 1). The control samples were tested
according to identical techniques in two laboratories (those of BH and
DVA). The control samples were tested late in the study, when initial
results from patients had suggested a high frequency of active
replication. Control samples were then intermixed with patient samples,
and the testing was done in blinded fashion by the same technicians
using the same reagents.
The method used to detect
the presence of HHV-6 in human peripheral blood lymphocytes was similar
to that previously described by one of us (SZS) (1). Briefly, peripheral
mononuclear cells were separated by Ficoll-Hypaque centrifugation at
1200 g for 45 minutes. The cells were collected, washed once in RPMI
1640 medium, placed in RPMI 1640 medium supplemented with 10% heat
inactivated fetal calf serum and penicillin-streptomycin, and incubated
at 37 degrees C for 24 hours under 5% CO2. Cells were then washed,
exposed to phytohemagglutinin (5 ug/mL) for 48 hours, collected by
centrifugation, and resuspended in media containing dexamethasone (5
ug/mL). The cell cultures were then observed daily.
We used modification of
the standard immunofluorescence technique to confirm infection with
HHV-6. Briefly, the patients' cells were fixed in ethanol, then
centrifuged and resuspended in RPMI 1640 medium containing 50% fetal
calf serum. The desired number of cells was placed on a slide, fixed for
30 minutes in cold methanol, and air-dried. The fixed cells were then
exposed to the desired dilution of test and control sera, followed by
exposure to a fluorescein-labeled secondary human antibody (Chemicon
International. Inc., Temecula, California). Test sera contained high
levels of antibody to HHV-6 and no detectable antibody to Epstein-Barr
virus or human cytomegalovirus. Control sera had no detectable antibody
to HHV-6 but did have antibody to Epstein-Barr virus or human
cytomegalovirus, or both, as determined by standard immunofluorescence
assays. The presence of infected cells was determined by the degree of
fluorescence relative to that seen in cell lines infected with
Epstein-Barr virus (B95-8 and Raji) and a cell line infected with human
A "positive bioassay" for
active HHV-6 infection was defined by the following findings (I): I)
After 4 to 8 days in culture, refractive "giant cells" developed that
increased in number and then underwent cytolysis, the cytopathic effect
typical of HHV-6; 2) these giant cells showed diffuse fluorescence
(typically not limited to either the nucleus or the membrane) when
incubated with HHV-6-positive serum and then with fluorescein-labeled
antibody; 3) the giant cells did not demonstrate fluorescence when
incubated with control sera (as defined above); and 4) normal-sized
cells that did not exhibit the characteristic cytopathic effect also did
not show fluorescence when incubated with HHV-6-positive serum. When
multiple longitudinal samples were obtained, a patient was considered to
have a "positive bioassay" if the number of positive results (as defined
above) was equal to or greater than the number of negative results.
Additional studies were
done in patients with positive bioassays to confirm the presence of
active HHV-6 infection. First, cord-blood lymphocytes were co-cultivated
with supernatant from six randomly selected patients who had a positive
bioassay. For purposes of control, cord-blood lymphocytes were also
co-cultivated with supernatant from three persons who had a negative
bioassay, and with supernatant from mock-infected HSB-2 cells, from
Epstein-Barr-virus-infected producer (P3HR-I) and nonproducer (Raji)
cell lines, and from cell-free human cytomegalovirus. Second,
fluorescein-labeled monoclonal antibodies developed by one of us (NB)
were used to stain cells from seven randomly selected patients who had a
positive bioassay; for purposes of control, three samples from patients
with a negative bioassay and uninfected HSB-2 cells were stained with
the same monoclonal antibodies in the lab- oratory of three of us (DVA,
SZS, and RCG). Three different monoclonal antibodies specific for
different HHV-6 (GS isolate) epitopes were used in the
immunofluorescence assay: 12B3G4, which recognizes a protein of 135 kDa;
6A5G3. which recognizes three glycoproteins of 54, 64 and 116 kDa; and
945D12, which recognizes proteins of 41 and 110 kDa (2). Third,
polymerase chain reaction was used to detect HHV-6-specific DNA
concurrently in cells from six randomly selected patients who had a
positive bioassay; for purposes of control, the study was also done in
cells from eight persons who had a negative bioassay. The specificity of
the HHV-6 probe was also tested by determining whether it hybridized to
cell DNAs (VERO and HEL) and other virus DNAs (human cytomegalovirus,
herpes simplex virus types 1 and 2, varicella-zoster virus, and
Epstein-Barr virus); and whether it produced a positive signal with a
reaction involving the HHV-6 oligonucleotide primers, buffer, and Taq
polymerase, but without a DNA template. The polymerase chain reaction
assays were done according to the method of Saiki and colleagues (3).
Briefly, reactions were conducted using an annealing temperature of 56
degrees C for 2.5 min, an extension temperature of 72 degrees C for 1.7
min, and a denaturation temperature of 94 degrees C for 1.7 min. A
series of 40 amplification cycles was used for each reaction.
Coded sera from 134 patients were tested for IgG
antibody reactivity against HHV-6-associated antigens by end-point
dilution enzyme-linked immunosorbent assay (ELISA) in the laboratory of
two of us (CS and RCG), according to previously described techniques
(4). These 134 patients were similar to the remaining 125 patients not
tested (see Table 2). Coded sera from 27 healthy control subjects from
the same community who were somewhat older but similar in gender
distribution to the patients (see Table l) were also tested.
Serologic test panels for
Epstein-Barr virus were done at least once in 249 of 259 patients (96%)
at Nichols Institute Immunology Reference Laboratory, San Juan
Capistrano, California. according to standard techniques (5, 6).
(Specimens from 29 patients and control subjects were tested in the
laboratory of the late Dr. Werner Henle at the Children's Hospital of
Philadelphia, and values were within one tube dilution of those observed
at the Nichols Institute.) Coded sera from 36 healthy control subjects
(mean age, 47.4 +/- 11.4 years: 56% female) from the same community were
also tested for antibodies to Epstein-Barr virus.
Testing for human
cytomegalovirus and Toxoplasma gondii was also done by standard
immunofluorescence assay at Nichols Institute.
Table 2. Characteristics
of Tested and Untested Patients *
Patients Patients P
Tested Not Tested Value †
HHV-6 replication assay
Mean age. y 39.5 +/-
11.2 38.3 +/- 12.6 > 0.2
70 67 > 0.2
College graduates, %
37 42 > 0.2
Bedridden or shut-in ‡. %
40 20 < 0.01
Chronic headaches ‡, %
93 79 < 0.01
Difficulty in sleeping ‡. %
44 56 < 0.01
Paresthesias ‡. %
44 56 < 0.01
HHV-6 serologic testing
Mean age, y 38.4 +/-
12.3 39.2 +/- 11.2 > 0 2
63 73 0.11
College graduates. %
41 36 > 0.2
Bedridden or shut-in ‡. %
21 29 < 0.01
Paresthesias ‡, %
30 48 < 0.01
Mean age, y 41.2 +/-
11.7 36.7 +/- 12.0 0.01
70 65 > 0.2
College graduates, %
43 36 > 0.2
Mean age, y 38.8 +/-
10.8 38.7 +/- 13.4 > 0.2
72 62 0.01
College graduates, %
43 35 0.22
Unable to work full-time ‡, %
67 37 < 0.01
Chronic headaches ‡, %
91 78 < 0.01
Chronic nausea ‡, %
63 38 < 0.01
Arthralgias ‡, %
80 61 < 0.01
Paresthesias ‡, %
75 47 < 0.01
HHV-6 = human herpesvirus type 6. MRI = magnetic resonance imaging.
Mean values are expressed +/- SD.
Alpha = 0.01 (see Tukey ).
Symptom or descriptor (out of a total of 190 evaluated) for which a
statistically significant difference was found between the patients
tested and those not tested.
Human Retrovirus Studies
Testing for antibodies to
human T lymphotropic virus type 1 (HTLV-1) was done using a conventional
ELISA (DuPont, Wilmington, Delaware). Testing for HTLV-I was done in 49
patients whose mean age (38.1 +/- 13.7 years), gender distribution (69%
female), and education (39% college graduates) were not significantly
different from those of patients not tested. Testing for antibodies to
human immunodeficiency virus (HIV) was done in 39 patients whose mean
age (39.5 +/- 11.0 years), gender distribution (51% female), and
education (44% college graduates) were not significantly different from
those of patients not tested. These serologic retroviral studies were
done in the laboratory of John L. Sullivan at the University of
Massachusetts Medical Center, as well as at Nichols Institute.
One of us (TF) attempted
to find evidence of a human retrovirus using heparinized blood from 14
patients. Peripheral mononuclear cells from the patients and from normal
persons (laboratory personnel) were isolated and stimulated using a
previously described method (7). After stimulation, patients' peripheral
mononuclear cells were either cultured in media alone or co-cultured
with normal stimulated peripheral mononuclear cells according to a
previously described method (8). Supernatants from these cultures were
tested for reverse transcriptase activity (7) over a 30-day period. The
primer used for the reverse transcriptase assay was oligo dT; the
template was poly rA. Both Mg++ and Mn++ were used as divalent cations,
because the reverse transcriptase from different known human
retroviruses has different cation dependency. The patients' cells were
established in culture only once. The supernatants were tested at 3-day
intervals for l month (approximately 10 tests for reverse-transcriptase
activity per patient over the 1-month culture period).
When percentages of patients and controls
with a certain clinical or laboratory finding were compared, we used the
chi-square or Fisher exact test. Intergroup comparisons of the
distributions of continuous variables were done using either t-tests or
Wilcoxon rank-sum tests (when distributions were non-normal). When a
large number of similar comparisons was made between two groups (such as
comparing the frequency of multiple symptoms), a P value for detecting
type I errors was chosen according to the method of Tukey (9). When
distributions were non-normal, we normalized them by log-transformation
(base 10). When multiple values for a variable were obtained for an
individual at different points in time, we calculated a mean value for
the variable after first normalizing the distribution (if necessary).
For example, a geometric mean titer for Epstein-Barr virus antibody was
calculated for each patient, as described previously (10).
Onset of Illness
Most patients experienced the onset of
illness between 1984 and 1986 (Figure 1), although in some cases the
illness had begun years before. No seasonal preponderance of cases was
"Clustering" of Cases
In the Tahoe group, several groups of
patients who had frequent close contact became ill within several months
of each other: 10 of 31 teachers at one local high school (at least one
student from the same school was similarly affected, but this student
chose not to participate in the study); 5 of 28 teachers and 3 students
at another local high school; 3 students and 1 teacher at a third high
school; and 11 employees at a casino. The spouses or sexual contacts of
six patients were similarly afflicted, and there were eight instances in
which at least one parent and one child both had the illness. Clinical
and laboratory findings in the "clustered" patients did not differ from
those of the larger patient group. Altogether, 101 of 183 patients (55%)
in the Tahoe group and 39 of 76 patients (51%) in the non-Tahoe group
stated that a close contact was similarly affected.
Symptoms and Signs
The two groups were generally similar
regarding symptoms and signs. In most patients, the chronic,
debilitating illness was of sudden onset, beginning with a "flu-like"
syndrome; 29% of the patients were regularly bedridden or shut-in. The
symptoms (Table 3) were chronic and were experienced on a nearly daily
basis in the months and years after the typically sudden onset of the
illness. These chronic symptoms constituted a new experience for most
patients: Very few of the patients reported these chronic symptoms in
the years before illness onset. For example, difficulty in concentrating
was experienced chronically by 3% of patients before the onset of the
illness but by 82% of patients after the onset of illness; depression
(as reported by patients) was experienced chronically in 6% of patients
before onset but by 68% after onset. Patients in the non-Tahoe group
were more frequently shut-in and had a slightly higher frequency of
headaches, adenopathy, arthralgias, paresthesias, and rash, but
generally the two groups were similar (Table 3). No statistically
significant differences were found when patients who experienced the
onset of illness before 1984 were compared with those who had a later
onset of illness. No statistically significant differences were found
between patients whose illness began abruptly with "flu-like" symptoms
and the few patients who experienced a more insidious onset, except that
abdominal pain was more frequent in the former group.
Twenty-two patients (8%) had evidence of
an acute neuropathic process, which generally occurred within several
weeks of the acute onset. Acute neurologic symptoms included primary
seizure disorder (7 patients); profound, transient ataxia of acute onset
(10 patients); and transient paresis (8 patients). Ataxia and paresis
were confirmed by neurologic examination (done by PRC and DLP) and
typically lasted 1 to 4 weeks; none of the patients sustained a
permanent deficit. The frequency of acute neurologic events was similar
in the Tahoe and non-Tahoe groups (see Table 3).
Standard Laboratory Tests
Laboratory tests generally yielded
unremarkable results. However, 31 of 172 patients (18%) had atypical
lymphocytosis (mean percentage of atypical cells, 6.5%). In addition, 21
of 81 patients (27%) tested had an antinuclear antibody titer of 1:20
(only 1 patient had a titer as high as 1:320); no patient had clinical
evidence of systemic lupus erythematosus. The frequency of these
laboratory abnormalities was similar in the Tahoe and non-Tahoe groups.
Cerebrospinal Fluid Examination
Of the six patients whose cerebrospinal
fluid was studied, one had pleocytosis (leukocyte count, 8 x 106/
L). Glucose and protein levels were unremarkable in all cases.
Oligoclonal bands were absent. No bacterial organisms were cultured from
any patient. In one instance, Epstein-Barr virus antibodies were
measured: Neither viral capsid antigen IgG nor viral capsid antigen IgM
was detected. Unfortunately, no cerebrospinal fluid was available for
HHV-6 isolation or serologic studies.
Magnetic Resonance Imaging of the Brain
Foci of high signal intensity on
T2-weighted images, typically punctate and occasionally larger patchy
areas, were seen in 113 of 144 patients (78%) but in only 10 of 47
matched healthy controls (21%) (P < 10-9) (Table 4). The
films were interpreted independently by two neuroradiologists (RB and
FAL), who agreed about the presence or absence of abnormal signal in 97%
of the patients. Representative images are shown in Figure 2. The
subcortical white matter was affected most often, but white matter
elsewhere in the central nervous system was also affected. A relation
was seen between the anatomic area affected and the clinical
presentation: One patient with ataxia had high signal intensity areas
involving the cerebellum (Figure 2, panel D), seven patients with visual
symptoms had high signal intensity areas involving the occipital cortex,
and one patient with paresis had a high signal intensity area involving
the contralateral internal capsule. In several cases, MRI studies were
repeated, and the scans showed that areas of high signal intensity
persisted even after symptoms resolved.
Lymphocyte Phenotyping Studies
The CD4/CD8 ratio was higher in each of
the two patient groups when compared with the control group (Tahoe
group, 3.11 +/- 1.6; non-Tahoe group, 3.19 +/- 1.40; control group, 2.30
+/- 1.00 [P < 0.003]). The higher CD4/CD8 ratios in the patient groups
were accounted for by both higher numbers of CD4+ cells (934.4 +/- 319.6
cells/mm' for the patients and 871.9 +/- 389.0 cells/mm' for the control
subjects; P = 0.07) and lower numbers of CD8+ cells (370.2 +/- 215.1
cells/mm3 for the patients and 452.0 +/- 257.6 cells/mm' for the control
subjects; P = 0.01). No significant differences were noted in the total
T-cell or B-cell number.
Table 3. Clinical
(N = 183) (N =
History of fatigue
Severity of the fatigue
Bedridden (can do virtually
nothing) 11 (6) 3 (4)
Shut-in (cannot do even light
or its equivalent or carry out
responsibilities) 32 (17)* 28 (37)*
Can work on only a part-time
basis 51 (28) 22 (29)
Can fulfill all home or work
responsibilities but is much more
easily fatigued from such
(no energy left for anything
else) 89 (49)* 23 (30)*
Chronic fatigue started suddenly with
a "flu," "cold," or virus
† 156 (85) 69 (91)
151 (83) 69 (91)
Headaches 149 (81) 71 (93)
concentrating 144 (79) 67 (88)
134 (73) 63 (83)
glands 130 (71) 65 (86)
sleeping 121 (66) 42 (55)
Anxiety 129 (70) 54 (71)
Depression or unusual mood
changes 122 (67) 55 (72)
pain 122 (67) 62 (82)
Cough 99 (54) 38 (50)
Recurrent fevers at
home 79 (43) 41 (54)
Nausea 88 (48) 47 (62)
83 (45) 39 (51)
appetite 70 (38) 27 (36)
Odd sensations in the
skin 73 (40)* 52 (68)*
Intermittent swelling of the
fingers 81 (44) 42 (55)
Diarrhea 60 (33) 32 (42)
Weight gain of more than 4.54 kg (10
Lb) 44 (24) 26 (34)
Eczema 37 (20)* 30 (39)*
54 (30) 33 (43)
Vomiting 29 (16) 9 (12)
Weight loss of more than 4.54 kg (10
Ib) 23 (13) 12 (16)
disorder 4 (2) 3 (4)
ataxia 7 (4) 3 (4)
paresis 3 (2) 5 (7)
Anterior cervical adenopathy
(enlarged or tender
nodes) 118 (66) 44 (64)
Posterior cervical adenopathy
(enlarged or tender
nodes) 91 (51) 36 (52)
Temperature of more than 37.6degrees
C 10 (6) 2 (3)
P < 0.01; threshold for significance given that more than 20 comparisons
were made (see Tukey ).
Characterized by at least two of the following symptoms: fever,
headache, myalgias, sore throat, earache, congestion, runny nose, cough,
diarrhea, and fatigue.
Results of the
Magnetic Resonance Imaging Studies of the Brain and of the Bioassay for
Replication of Human Herpesvirus Type 6 in the Tahoe Group, the
non-Tahoe Group, and the Control Group*
Non- Control P
Tahoe Group Value †
n/n (%) ) ----------]
Hyperintense signal in
white matter on MRI
study of the brain 71/91 (78)
42/53 (79) 10/47 (21)
Positive bioassay 45/71 (63)
34/42 (81) 8/40 (20)
The Tahoe group comprised patients from the Lake Tahoe area near the
Nevada-California border: this patient group was also referred to as the
"epidemic" group because most of the patients had many contacts who also
became ill. The non-Tahoe group comprised patients from outside the Lake
Tahoe area: this patient group was also referred to as the "endemic
group" because patients did not have many contacts who became ill. The
control group comprised healthy persons. MRI = magnetic resonance
† P value for the
comparison of two patient groups with the control group.
Assays for Active Replication of Human Herpesvirus Type 6
Two hundred and thirty-five specimens
were obtained from 113 patients. A positive bioassay for HHV-6 (Figure
3) was seen in 70% of the patients and 20% of the control subjects (P <
10-8) (see Table 4). Most of the assays in 27 of the 34 patients (79%)
who had serial testing for HHV-6 indicated active replication of HHV-6.
When, later in the study, samples from control subjects were intermixed
with those from patients, the rate of positivity among patients was as
high as it had been before control samples were intermixed, indicating
that the earlier reading of patient samples was accurate.
Confirmatory studies for HHV-6
replication were also done. Co-cultivation of cord-blood lymphocytes
with supernatant from primary cultures yielded a positive cytopathic
effect in each of the six patients tested; no cytopathic effect was seen
in any of the controls. In two patients showing a positive cytopathic
effect in the primary co-cultivation study, secondary co-cultivation
studies using supernatant from the presumably infected cord-blood
lymphocyte cultures were done. The characteristic cytopathic effect was
seen in the new cord- blood lymphocyte cultures in each of the two
patients tested. Monoclonal antibody studies were positive with all
three monoclonal antibodies in all seven patients studied, confirming
that HHV-6-specific antigens were present in patients who had a positive
bioassay. Monoclonal antibody staining was not observed in the controls.
Polymerase chain reaction studies were shown to be specific for HHV-6,
as described in the Methods section; the studies were positive in all
six patients who had a positive bioassay and in none of the controls,
confirming that HHV-6-specific nucleic acid sequences were also present
in patients with a positive bioassay. As reported elsewhere, Southern
blot examination was also done in three patients (11), which confirmed
the presence of HHV-6 in all three instances.
Of the 113 patients studied for HHV-6
replication, 84 (74%) underwent MRI study; 68 of these 84 patients (8l%)
had MRI abnormalities, a proportion similar to that observed for all
patients who had MRI study (79%). Patients with abnormalities were not
more likely to have evidence of actively replicating HHV-6 (P = 0.6).
Median optical density values for the
HHV-6 IgG ELISA in patients and control subjects were 1905 and 1288,
respectively (P = 0.08, one-tail). Ninety-three percent of patients were
seropositive for Epstein-Barr virus; only two patients had serologic
evidence of primary Epstein-Barr virus infection. The reciprocal
geometric mean titers (+/- SD) for the Tahoe and non-Tahoe groups were
significantly higher than those of the control group for Epstein-Barr
virus viral capsid antigen IgG (138.0 +/- 2.6 for the Tahoe group, 154.9
+/- 3.0 for the non-Tahoe group, and 67.6 +/- 4.4 for the control group;
P < 0.0001); and for early-antigen R antibody (40.7 +/- 2.6 for the
Tahoe group, 30.9 +/- 2.2 for the non-Tahoe group, and 12.6 +/- 6.0 for
the control subjects, P < 0.0001); there were no statistically
significant differences in values for viral capsid antigen IgM,
early-antigen D antibody, and Epstein-Barr nuclear-antigen antibody.
Of 92 patients tested, 45 (49%) had
cytomegalovirus IgG antibody levels of more than 1:20 and 10 had low
levels of cytomegalovirus IgM antibody. Low levels of IgG and IgM
antibody to Toxoplasma gondii were found in 7 of 29 (24%) and in 1 of 11
(9%) patients tested, respectively.
Human Retrovirus Studies
Serologic test results for antibodies to
HTLV-I and antibodies to HIV were negative in the patients tested. The
14 culture supernatants tested were negative for reverse transcriptase
activity by both assays (Mg++ and Mn++).
The illness we have observed shares many features with the entities
variably called "postinfectious" or "postviral" fatigue syndrome (12);
myalgic encephalomyelitis, Royal Free disease, or Icelandic disease
(13-19); primary fibromyalgia (or fibrositis) (20-22); chronic
mononucleosis (23-25); chronic active Epstein-Barr virus infection
syndrome (26-28); and the chronic fatigue syndrome (29, 30). Perhaps
these differently named entities are the same illness and share a common
etiologic agent; perhaps they are clinically similar but separate
illnesses, with each triggered by a particular and different etiologic
agent; or perhaps these entities represent one illness that can be
triggered by various factors and that manifests different clinical
features in different patients (perhaps because of host factors) but
causes chronic fatigue and certain common pathophysiologic features in
all patients. We are more inclined to the last view.
We cannot say precisely if the illness we witnessed from 1984 to 1986
meets criteria for chronic fatigue syndrome because the case definition
for that illness developed in 1988 (30) includes some symptoms (for
example, a greater than 50% reduction in function) that we did not
explicitly ask the patients about, using the same language.
Nevertheless, most patients probably would have met the case definition
for chronic fatigue syndrome because the illness began suddenly in 87%
of patients, the median duration of the illness at time of study entry
was 1.3 years, 57% of patients were bed- ridden, shut-in, or unable to
work full-time, and most had the key symptoms required by the case
definition (see Table 3).
For most patients in our study, the illness consisted largely of
subjective and nonspecific symptoms, and the results of common
laboratory tests were often normal. Initially, it was unclear to us
whether the patients were experiencing an organic illness or the somatic
manifestations of a psychological illness. The several objective
neurologic, immunologic, and virologic findings make a diagnosis of
purely psychological illness unlikely, although it remains possible that
these findings are biologic concomitants of primary psychiatric illness.
Studies of that possibility are in progress.
We frequently saw areas of abnormal signal intensity in the white matter
of the central nervous system. Without studies of central nervous system
tissue, we cannot be certain about the meaning of these findings.
Progress in characterizing white matter abnormalities using magnetic
resonance imaging has been severely inhibited by the absence of tissue.
In normal persons, for example, hyperintense signal on T2-weighted
images is seen anterior to the frontal horns (31). Similar signal
characteristics are seen in the periventricular white matter lesions of
patients with multiple sclerosis; because no signal feature can be used
to differentiate these MRI signal abnormalities, only their spatial
distribution identifies them. Many different diseases in addition to
multiple sclerosis are associated with areas of high signal intensity
caused by white matter edema or demyelination, or both: Alzheimer's
disease; vascular dementia (Binswanger disease, lacunar state,
multi-infarct dementia); normal pressure hydrocephalus; metastatic
disease; trauma; leukodystrophies; secondary Wallerian degeneration from
any brain damage; and post-irradiation, post-chemotherapy, toxic, or
metabolic leukoencephalopathies. Viral infection (for example, with the
retro-viruses HIV  or HTLV-I ) also commonly produces similar
white matter changes. As was the case in our study, such punctate areas
of high signal intensity can also be seen in apparently healthy persons
of all ages.
The clinical significance of these "incidental" areas of high signal
intensity in the white matter is not known (34-37); their prevalence
appears to rise with age and with the presence of cerebrovascular risk
factors. Particularly ambiguous is the interpretation of punctate areas
of high signal intensity seen mostly in the subcortical white matter.
The distribution of this finding corresponds anatomically to the
cerebrospinal-fulfilled perivascular (Virchow-Robin) spaces that can be
seen on high-resolution magnetic resonance images. The enlarged Virchow-Robin
spaces may be a normal variant or may be the result of either
cerebrospinal fluid penetration into ischemic white matter or cellular
infiltration within the dilated perivascular spaces as part of an
inflammatory process (38, 39). The areas of high signal intensity seen
in our patients and control subjects varied in size and location and may
arise from different causes. The much greater frequency of high signal
intensity in the white matter of both the Tahoe and non-Tahoe groups
than in the control group (P < 10-9), the presence in some
patients of larger and deeper white matter lesions, and the correlation
between the anatomic area of involvement and clinical symptoms all
suggest that the patients were experiencing a genuine but as yet
undefined pathologic process.
We cannot judge how generalizable the MRI findings might be to other
patients with a similar syndrome. In our preliminary studies of patients
with chronic fatigue syndrome from New England, MRI abnormalities were
seen less commonly (40% to 50% of patients) than in this patient group.
We do not recommend routine use of MRI studies in patients with
suspected chronic fatigue syndrome; the value of the test first needs to
be assessed in other populations. We and others are currently evaluating
magnetic resonance imaging and other neuroimaging techniques in chronic
Immunologic (lymphocyte phenotyping) studies revealed a significantly
increased CD4/CD8 ratio in each of the two patient groups when compared
with the control group (P < 0.003), because patients had both a higher
number of CD4+ T cells and a reduced number of circulating CD8+ T cells.
Landay and colleagues (40) have also found a reduced number of CD8+
cells in clinically similar patients, although some studies of
clinically similar patients have not found a similar increase in the
CD4/CD8 ratio (22, 40, 41). We did not examine T-cell subsets, but Gupta
and colleagues (41) as well as Straus and Strober (Unpublished data)
studied patients with an apparently similar syndrome and reported
elevated numbers of activated CD4+ cells. Klimas and coworkers (22) and
Landay and coworkers (40) have found an elevated number of activated
CD8+ cells, decreased numbers of CD4+ CD45RA+ "suppressorinducer" cells
(22), and decreased numbers of CD8+ 11b+ "suppressor" cells (40). We did
not measure T-cell function, but Klimas and colleagues (22), Murdoch
(42), and Lloyd and colleagues (43) have found evidence of cutaneous
anergy or T-cell dysfunction, or both, as reflected by conventional
mitogen stimulation assays, in patients with a similar chronic illness.
Gupta and colleagues (41) found T-cell dysfunction in response to
challenge with soluble antigens. Increased B-cell numbers have been
reported in some patients with chronic fatigue syndrome (22, 44) but not
in others (40, 41). An increase in the B-cell subset (CD20+ CD5+) that
may be dedicated to the production of autoantibodies (45) has been
reported by Klimas and colleagues (22). Increased numbers of activated
monocytes have been observed (41). We have reported previously that
natural killer cell phenotypic and functional abnormalities are also
present in patients with chronic fatigue syndrome: Such patients show
reduced numbers of the NKH1+ T3 - subset, and their natural killer cells
demonstrate defective cytolytic activity against target cell lines (46).
Other investigators have also found defective natural killer cell
function (22, 47-49). Indeed, defective natural killer cell function may
be the most commonly reported abnormality in patients with chronic
fatigue syndrome. Taken together, the controlled studies cited above and
many others summarized elsewhere (50) seem to indicate an immune system
chronically responding to a "perceived" antigenic challenge.
The virologic studies we have done thus far indicate that two human
herpesviruses may be actively replicating more often in patients than in
control subjects. The first is Epstein-Barr virus, and the indirect
evidence for its active replication in patients with chronic fatigue
syndrome comes from the Epstein-Barr virus antibody profiles, which are
characterized by higher levels of IgG antibody to both viral capsid
antigens and early antigens. The second such virus is HHV-6, which was
recently discovered in one of our laboratories (RCG) (1, 51, 52). With
HHV-6, the evidence of active viral replication is direct: When
lymphocytes from the two patient groups were placed in primary cell
culture, a cytopathic effect typical of HHV-6 was seen in 70% of
patients but in only 20% of control subjects (P < 10-8) (see
Table 2 and Figure 3). The use of three different monoclonal antibodies
specific for HHV-6 epitopes confirmed that HHV-6 protein was present in
cells exhibiting cytopathic effect, and the application of polymerase
chain reaction using probes specific for HHV-6 nucleic acid sequences
also confirmed the presence of HHV-6. The only other known human virus
that might also have been detected with these assays was the closely
related, recently discovered human herpesvirus type 7 (53). Finally, in
every instance, supernatant from the primary cell culture produced the
same cytopathic effect in cord-blood lymphocytes, demonstrating that a
transmissible agent was indeed present in the patients' lymphocytes and
that this agent was HHV-6.
We do not know how generalizable these findings may be. Several other
groups have found elevated antibodies to Epstein-Barr virus (23, 26-28).
After our first report (54), several groups reported elevated antibody
levels to HHV-6 (40, 55-57) in patients with a similar illness. As for
our finding of active HHV-6 replication in patients' lymphocytes,
although one group has been unable to induce active replication of HHV-6
in the lymphocytes of healthy persons (57), others (58, 59; Gillespie D.
Personal communication; Yamanishi K. Personal communication) have been
able to do so with a frequency similar to that which we report. Like us,
some investigators (59; Gillespie D. Personal communication) also have
found evidence of inducible active HHV-6 replication in patients with
chronic fatigue syndrome. At this time, because the assays are difficult
to do and because their value has yet to be tested in other populations,
we do not recommend the routine use of HHV-6 testing in patients with
suspected chronic fatigue syndrome.
It appears that most patients acquire primary infection with HHV-6 early
in life (4). Therefore, the active replication of HHV-6 seen in most
patients probably represents reactivation of an old, latent infection
rather than a new, primary infection with the virus. This reactivation
of HHV-6 could be nothing more than an epiphenomenon, secondary to
immune dysfunction or transactivation by another unrecognized virus, and
could have no relation to patients' symptoms. Alternatively, whatever
the mechanism of reactivation, HHV-6 might contribute to producing the
morbidity associated with this illness. Such a hypothesis is plausible
because HHV-6 is tropic for T cells; B cells; and glial, neuroblastoma,
and intestinal cell lines (60, 61).
Our study had several limitations. We could not systematically survey a
random sample of the denominator population in the community; hence, we
could not assess disease prevalence nor could we examine more closely
the possible clustering of cases. Magnetic resonance imaging lymphocyte
phenotyping, and the assays for HHV-6 replication were not done in all
259 study patients; nevertheless, the large samples tested were
sufficiently similar to the total group (see Table 2 and the Results
section) that the striking differences between patients and healthy
control subjects are unlikely to be explained by a sampling bias. The
MRI findings were interpreted independently by two neuroradiologists,
with very high agreement. However, because films from patients could be
distinguished from those of control subjects by the logos on each image,
interpretation was non-blinded.
We do not argue that these observations identify the cause of this
illness, nor can we be sure our findings will be confirmed in other
populations of patients with apparently similar illnesses. Indeed, as
stated earlier, we think that this probably is a heterogeneous illness
that can be triggered by multiple different genetic and environmental
factors (including stress, toxins, and exogenous infectious agents), all
of which can lead to immune dysfunction and the consequent reactivation
of latent viruses (29). Several different exogenous and endogenous
infectious agents may be involved in this illness, acting singly in some
cases and collaboratively in others. In addition to HHV-6 and
Epstein-Barr virus, the enteroviruses (62-65), Borrelia burgdorferi
(66), and other infectious agents (67) may be involved in some cases.
Preliminary evidence suggests that a novel retrovirus may be involved in
this illness (68); at least one other retrovirus, HIV, is known to act
synergistically with HHV-6 (69, 70). In our study, the apparently
"epidemic" Tahoe cases may have been triggered by different agents than
the apparently "endemic" non-Tahoe cases; however, the remarkably
similar clinical and laboratory findings in the two patient groups
suggest a final common pathogenetic pathway involving immune
dysregulation. We hope that further investigation generated by these
observations may lead to a better understanding of this often
authors thank S. Baker, E. F. Cook. K. Cullen, K. Dibvig, S.
DeLaFerriere, N. Fiebach, E. Gebhardt, V. Haughton, D. Holt, M. Jones.
A. Kinter, E. Keiff; J. Kornish. C. Lawyer, J. Lee, P. Levine. R.
Lissner, C. Martin, R. Schmidt. H. Streicher, J. L. Sullivan, D. Swain,
R. S. Thomas, D. Watson. R. A. Weinberg, and D. Willitts for their help
with the study or their advice: and R. Honess for provision of
HHV-6-specific oligonucleotide primers and the HHV-6 DNA plasmid, pHD5.
Grant Support: By the National Institutes of Health
(grants RO1A1 26788, RO1A127314. UO1A132246. 2PO1CA41167 and
SK04NSO1083); and by funds from M. Palevsky. S. Harris, the S. Sydney
DeYoung Foundation, the Minann Foundation, the Pioneer Foundation, the
Rowland Foundation, and Sierra Research Institute. Dr. Buchwald was
supported by a fellowship from the Henry J. Kaiser Family Foundation and
by a Young Investigator Award from the National Alliance for Research on
Schizophrenia and Depression.
Requests for Reprints:
Anthony L. Komaroff, Division of General Medicine, Department of
Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston MA
Current Author Addresses:
Dr. Buchwald: Harborview
Medical Center, General Internal Medicine. 325 Ninth Avenue, ZA-60,
Seattle, WA 98104-2402.
Dr. Cheney: 10620 Park
Avenue, Suite 234, Charlotte, NC 282IO.
Dr. Peterson: 865 Tahoe
Boulevard, Suite 306, Incline Village. NV 98450.
Dr. Henry: Washoe County
Sheriffs Division. Forensic Science Division, 911 Parr Boulevard, Reno
Ms. Wormsley: Cytometry
Associates, 11575 Sorrento Valley Road, #204, San Diego, CA 92121.
Ms. Geiger: l737
Broadview Lane, P416, Ann Arbor, Ml 48105.
Dr. Ablashi: Laboratory
of Cellular and Molecular Biology, Building 37, National Cancer
Institute, National Institutes of Health, Bethesda, MD 20892.
Huntington Memorial Hospital, University of Southern California, AIDS
Research Laboratory, 744 Fairmont Avenue. Pasadena. CA 91105.
Drs. Saxinger and Gallo:
Laboratory of Tumor Cell Biology, Building 37, Room 6B10, National
Cancer institute, National Institutes of Health, Bethesda, MD 20892.
Dr. Biddle: Stockton
Radiology Medical Group, 1617 North California Street. California
Medical Center, Suite 1A, Stockton. CA 95204.
Drs. Kikinis. Jolesz, and Komaroff:
Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115.
Dr. Folks: Division of
Virology, Centers for Disease Control, 1600 Clifton Road, Atlanta GA
Department of Microbiology, University of Kansas Medical Center, Kansas
City, KS 66I03.
Dr. Peter: Specialty
Laboratories, Inc., P.O. Box 92722, Santa Monica, CA 90009.
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Annals of Internal
Medicine 1992 Volume 116
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