Double-blind , randomized study of the effects of influenza vaccination on the specific antibody response and clinical course of patients with chronic fatigue syndrome

Division of Infectious Disease, Department of Medicine; Sleep Disorders Program, Department of Psychiatry, University of British Columbia and Vancouver Hospital and Health Sciences Centre, Vancouver, British Columbia Correspondence and reprints: Dr HG Stiver, Division of Infectious Disease, D-floor, 2733 Heather Street, Vancouver, British Columbia V5Z 3J5. Telephone 604-875-4146, fax 604-875-4013, e-mail gstiver@interchange.ubc.ca Received for publication May 14, 1999. Accepted November 26, 1999 KM Sleigh, DG Danforth, RT Hall, JA Fleming, HG Stiver. Double-blind, randomized study of the effects of influenza vaccination on the specific antibody response and clinical course of patients with chronic fatigue syndrome. Can J Infect Dis 2000;11(5):267-273.


BACKGROUND
Chronic fatigue syndrome (CFS), also known as 'myalgic encephalomyelitis' (ME), is a disorder characterized by the new onset of a persistent or relapsing fatigue that fails to resolve with bedrest and that significantly impairs daily activity for six months or longer.Conditions that may account for the patient's symptoms must be excluded before CFS may be diagnosed (1).Three definitions of CFS exist in the literature, but all include the preceding description of debilitating fatigue.The Centers for Disease Control and Prevention (CDC, Atlanta, Georgia) definition stipulates that patients must also experience at least six symptoms and two signs, or eight symptoms of CFS.Signs and symptoms include fever, pharyngitis, cervical adenopathy, myalgias, postexertional fatigue, headaches, neuropsychological disturbances and sleep disorder (1).The Australian definition does not require that patients experience any particular sign or symptom but specifies the presence of neuropsychiatric impairment and/or abnormal cell-mediated immunity (2).The Oxford definition is the most lenient, requiring only the existence of debilitating fatigue (3).
In a community-based population, the point prevalence of CFS was reported to be 98 to 267 cases/100,000 (4).CFS sufferers are often unable to work and are dependent on disability insurance from private insurers and government pension plans.Conditions that seem to exacerbate the illness are any undue physical or mental stress, such as an acute viral illness or psychologically stressful event.These may result in a setback that may last several months, further compounding the disability.
The etiology of CFS continues to be the subject of vigorous debate.CFS has been attributed to the reactivation of latent infectious agents and/or immune dysfunction (although evidence suggests only in vitro immune alterations) (5)(6)(7)(8)(9).The high rate of pre-and comorbid psychiatric illnesses in CFS patients has prompted some theorists to advance a psychological basis for the disorder (10)(11).Demitrack (12) suggested that the phenomenological overlap between CFS and primary psychiatric illnesses reflects the existence of a shared, final common biological pathway, the hypothalamic-pituitary-adrenal axis (HPA), which may become disturbed by a variety of infectious or noninfectious pathophysiological antecedents.
In our experience, some patients are reluctant to receive common preventive agents such as vaccines for fear of exacerbating CFS symptoms.To provide objective data on immunization in CFS patients, we investigated the effect of commercially available influenza vaccine on the following: the specific antibody response; the rate of early post-immunization side effects; and any effects of the immunization on the clinical course of CFS for the following six weeks.Our null hypothesis was that there would be no difference in antibody responses for CFS vaccinees compared with healthy vaccinees and no difference in the clinical course of patients with CFS who received vaccine compared with CFS patients receiving the placebo immunization.

PATIENTS AND METHODS
Patients: Because of the difficulty in demonstrating a fourfold antibody rise in individuals with higher pre-existing titres,

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Can J Infect Dis Vol 11 No 5 September/October 2000 prospective participants who had received an influenza immunization within two years of study entry were excluded.Individuals who were allergic to eggs were also excluded.The appropriate ethics committee approval was obtained, and participants provided signed, informed consent.Two samples were recruited: CFS patients who were randomly selected to receive immunization (n=19) or placebo (n=21); and healthy volunteers (n=21).CFS group: Outpatients who attended the CFS clinic were eligible to participate if they were diagnosed with CFS as defined by CDC criteria (1).A total of 107 patients were contacted by telephone.Fifty-two patients refused to participate or were deemed ineligible.Of the remaining 55 patients, 15 were excluded after signing informed consent.This group included eight patients who met the criteria for psychiatric exclusion, six patients were unable to make repeated visits to the clinic, and one patient was diagnosed with asthma as a cause of chronic fatigue.No differences were found between ineligible or excluded patients and the final CFS sample regarding the following demographics: age (P=0.58;t-test), sex ratio (P=0.77;c 2 ) and duration of CFS symptoms (P=0.71;t-test).
Control group: Twenty-five age (within three years) and sexmatched healthy volunteers who responded to advertisements published in various institutional newsletters were recruited for the comparative assessment of antibody response and immediate untoward effects of influenza vaccination.Twenty-one returned for the three week, postimmunization blood sampling.These persons were employees at the Vancouver Hospital, the University of British Columbia and Simon Fraser University, Vancouver, British Columbia.

Methods -CFS group:
All potential CFS patients underwent a history and physical examination.After demographic data and information on concomitant medication use were collected, a psychiatric assessment was performed using the Beck Depression Inventory (BDI) (13) and the Computerized Diagnostic Interview Schedule (CDIS) (14).After reviewing the CDIS responses, a psychiatrist interviewed patients to verify data generated by the CDIS.
To assess the effects of vaccination, data were collected on pre-and postimmunization health status and CFS symptoms.In addition, all patients maintained a seven-day diary of injection-related side effects during the week following immunization.All CFS patients returned self-administered questionnaires weekly by mail, using prestamped, addressed envelopes.Due to the cognitive impairment associated with CFS, participants were given a nine-week calendar identifying office visit dates and when each questionnaire was to be completed.Questionnaires were staggered throughout the study to reduce fatigue and measurement error.Despite the heavy time and concentration burden imposed by the questionnaires, patients completed the following questionnaires with few omissions: • The Sickness Impact Profile (SIP) ( 15) is a 136-item self-report questionnaire that measures sicknessrelated physical and psychosocial dysfunction experienced by respondents in the preceding three months.Patients completed the SIP preimmunization and at three and six weeks postimmunization.
• The Symptom Check List 90 (SCL-90) ( 16) is a 90-item instrument that assesses the number and severity of psychological and physical symptoms.The SCL-90 was completed before immunization and weekly for six weeks postimmunization.
• The BDI ( 13) is a reliable and valid scale that describes 21 symptoms of depression.Subjects were instructed to fill out the BDI preimmunization and weekly for six weeks after immunization.
• The Visual Analogue Scale for Fatigue (VAS-F) (17) uses an 18-item, nonsegmented analogue scale to measure fatigue and energy.Reliability for the fatigue and energy subscales in a group of sleep-disordered patients was calculated as 0.96 and 0.95, respectively (17).The instrument has demonstrated excellent concurrent validity (17).The VAS-F was administered for seven consecutive days preimmunization, seven consecutive days postimmunization, and then once a week for four weeks.

•
The St Mary's Hospital Sleep Questionnaire (SMHSQ) ( 18) is a 14-item instrument that evaluates a respondent's sleep and early morning behaviour for the preceding 24 h.Using Kendall's tau, test-retest reliability for the items ranged between 0.70 and 0.96 (18).Participants completed the SMHSQ each day for one week before and immediately following immunization, then each day for one week for weeks three and six after immunization.
• The CFS-related Symptom Time/Severity Questionnaire (CFS-SS) was developed by the authors to monitor patients' progress in the CFS clinic.Using a Likert scale, the CFS-SS provided a comparative semiquantitative measure of maximum symptom severity and duration between the two CFS treatment groups.The CFS-SS was completed weekly for three weeks before immunization, then weekly for six weeks after immunization.
• To measure early vaccine side effects, the Vaccine Evaluation Centre Questionnaire (VECQ) was used.Patients were given a thermometer and asked to measure their oral temperature four times daily for seven days postimmunization.They were also asked to record any immunization-related symptoms and/or localized skin reactions each day.Serum sampling: Immediately before immunization and three weeks after immunization, a 10 mL specimen of blood was collected from each patient and allowed to clot.Sera were tested for the presence of hemagglutination-inhibiting (HI) antibodies against the homologous antigens using a standard protocol, except nonspecific inhibitors were removed by kaolin treatment of sera rather than receptor-destroying enzyme neuraminidase (19).All sera were tested in pairs, and any twofold rises in antibody were retested twice, and the 'two of three' concurrent values applied in the analysis of data.No other measurements of immunological function were made.Control group: Blood was obtained for antibody testing and all participants were immunized with 0.5 mL of Fluviral S/F (as described above) and instructed to complete the VECQ and record their temperatures four times a day for one week.

Immunization
Participants returned to the hospital to have blood drawn for antibody testing three weeks postimmunization.Laboratory analysis of the serum was identical to the procedure used for the CFS group.Statistical analyses: Baseline antibody titres were compared using the Mann Whitney U test.Comparison of the numbers of patients with fourfold titre rises were analyzed with the c 2 test, and magnitude of titre rise was analyzed using the two sample t-test.
Temperature data derived from the VECQ forms were expected to be normally distributed and were analyzed using ANOVA.Symptom scores were non-normally distributed, and the Kruskal Wallis one-way ANOVA and Mann Whitney U tests were used to compare the values between the groups.Analysis of covariance was used to analyze the function and severity questionnaire data.The postinjection mean level was compared between the vaccine and placebo groups while adjusting for the pretest mean.Using Cohen's power tables, it was calculated that, in terms of overall group differences and group profiles, the study could detect a large effect size with a power of 0.70 (20).The effect size is the relative difference in the means between two comparisons divided by the standard deviation.A large effect size suggests a clinically significant difference and is demonstrated when the scores of two populations are highly divergent.

Sample:
The CFS sample was almost exclusively Caucasian (97%), and mostly composed of women (76%).Patients' ages ranged from 18 to 56 years, with a mean ± SD of 39± 10.On average, patients had experienced CFS symptoms for 47± 29 months.The mean age of illness onset was 36 years.These demographics are comparable with those reported by Ho-Yen and McNamara (21) in an analysis of data from several self-referral studies.Although statistically equivalent in age (P=0.13) and illness duration (P=0.63), the CFS treatment groups differed in medication use.Ten CFS vaccinees reported taking antidepressants versus four patients in the CFS placebo group.As well, five CFS vaccinees used sedatives, while only two patients in the CFS placebo group reported using sedation.
The healthy volunteers and the CFS vaccinees were well matched demographically.The average age of the controls and CFS vaccinees was 40 years, and no significant differences were found when the ages of the men (P=0.09,t-test) and women (P=0.47,t-test) in the CFS and control groups were compared.No individuals in either CFS group or the healthy volunteer group were taking, or had recently taken, immunosuppressive medication.volunteer and CFS placebo groups, respectively.The three placebo patients who developed fourfold rises were almost certainly infected with a wild type strain, and, therefore, it must be assumed that the antibody response for a similar proportion of the vaccinees may have been due to wild type incidental infection.However, the review of symptoms in the three groups failed to uncover any patients with a typical influenza illness during the postimmunization period.The baseline prevaccination reciprocal geometric mean titres (GMT) for each vaccine antigen comparing the two groups of vaccinees, healthy versus CFS were: 13.5 versus 23.9 for A/Texas/H 1 N 1 (P=0.007,Mann Whitney test), 10.4 versus 12.9 for A/Beijing/H 3 N 2 (not significantly different), and 9.1 versus 11.9 for B/Panama (not significantly different).

Antibody response to vaccination
There was a significantly greater magnitude of antibody titre rise in the healthy vaccinees compared with the CFS vaccinees except for B/Panama.The increase in GMT ± SD comparing healthy controls and CFS vaccinees was 33.1± 2.1 and 14.3± 1.86 (P<0.001,two sample t-test), respectively, for A/Texas/H 1 N 1 ; 39.4± 2.8 versus 26.70± 2.5 (P<0.001) for A/Beijing/H 3 N 2 ; and 5.6± 2.1 versus 5.9± 0.03 for B/Panama.

Immediate side effects from vaccination:
Comparison of the postimmunization symptom scores for the three groups (Table 1) revealed a striking and statistically significant increase in the values for the 25th, 50th, and 75th percentiles for the CFS groups over the healthy volunteers group.However, there was no statistical difference in immediate side effects between CFS patients who received influenza vaccine and those who received a saline injection.Similarly, there was no significant difference in the average highest daily temperature, an objective measurement, among the three groups.

Effects of vaccination on the course of CFS:
Patients completed a battery of questionnaires to measure the effects of vaccination on the course of CFS.Using each patient as his or her own control, comparisons of the preimmunization mean values with the mean values recorded over the six weeks after injection revealed that only the SMHSQ demonstrated statistically significant differences between the CFS vaccinees and placebo groups, with the placebo group experiencing higher scores for sleep latency (length of time to fall asleep at night).However, this finding likely reflects differences that were shown to exist between the two groups at baseline and may be attributable to the increased use of antidepressants and sedatives in the CFS vaccination group.

DISCUSSION
This study is the first to examine the effect of immunization on the function of patients with CFS in a double-blind protocol.Influenza immunization appears to be safe, with no more untoward early side effects than a placebo injection.However, CFS patients in both the placebo and immunized groups reported a number of constitutional symptoms in the first week postimmunization.Because the VECQ measures immunization-related effects, such as malaise and muscle aches, which are also associated with CFS, it is difficult to determine whether these symptoms could be attributed to immunization or CFS.Because there were no significant differences between the CFS placebo and vaccine groups in terms of symptom scores, we concluded that few if any of the postimmunization complaints in the CFS vaccinees were caused by the vaccine.This is supported further by the lack of any difference in highest daily mean temperature for the week.
The immunizing response to the three vaccine antigens studied was normal in patients with CFS.It has been suggested, based on laboratory studies (6-9), that CFS is a condition of immune unresponsiveness or dysregulation.This has Can J Infect Dis Vol 11 No 5 September/October 2000 271 Influenza vaccination and chronic fatigue syndrome  led to the term chronic fatigue immune dysfunction syndrome (CFIDS).Our data indicate a blunted humoral immune response to the influenza A antigens and less so to B/Panama.However, most of the CFS vaccinees mustered a fourfold HI antibody rise to at least a titre of 1:32, which has been correlated with protection against wild type infection (22,23).It is unlikely that the blunted responses of the CFS vaccinees were related to antidepressant use.Selective serotoninreuptake inhibitors were the preferred antidepressant agents used by the CFS vaccinees, and the selective serotoninreuptake inhibitor, fluoxetine, has been found to normalize levels of cytotoxic T cells and natural killer cell cytoxicity in a group of CFS patients treated for eight weeks in an uncontrolled trial (24).
In a previous study, differences were found in the cellular and humoral responses of 20 CFS patients fulfilling CDC criteria compared with 20 age-and sex-matched controls (8).CFS patients demonstrated differences in cell-mediated immunity, including a decreased response to soluble antigens.The humoral antibody titre response at two weeks postimmunization to a primary pneumococcal polysaccharide antigen was reported to be decreased in six of 20 CFS patients, but the response in the controls was not reported for comparison.The antibody response to diphtheria and tetanus protein antigens (anamnestic response to a booster dose of diphtheriatenanus) was reportedly normal, although data in patient and control groups were not shown.
Stress may account for differences in immune functioning between CFS patients and healthy people.Studies have suggested that CFS patients are vulnerable to stress.Researchers found that CFS patients experienced more loss-related life events 12 months before CFS onset than healthy control groups (25), attributed the onset of the disorder to stressful events and/or lifestyles (26), and linked exacerbations of CFS symptoms to stressful events (10,12,27).In addition, CFS has been shown to compromise social functioning and reduce patients' social support networks (28,29).
In two samples, Glaser et al (30) demonstrated differences in cellular and humoral immune responses to vaccines, which the authors attributed to stress.In an older population consisting of caregivers of Alzheimer's patients, subjects inoculated with commercially available, inactive influenza vaccine showed a poorer antibody response and more rapid decline in the virus-specific T cell response over time than matched controls.Similar findings were reported in a group of medical students who were inoculated with hepatitis B vaccine injections following a three-day examination period.Twelve of the 48 medical students seroconverted after the first injection.These students were found to fall into the lower stress/low anxiety group.In addition, students who reported greater social support and lower anxiety and stress demonstrated a higher antibody response to the vaccine and a more vigorous T cell response to hepatitis B surface antigen.A variety of mechanisms have been hypothesized to account for the effect of stress on the immune system (31,32).
We observed two patients in the placebo group with fourfold rises in antibody to A/Beijing/H 3 N 2 and one with a fourfold rise to A/Texas H 1 N 1 .Because the study was conducted in the fall of 1993, and both H 3 N 2 and H 1 N 1 wild type viruses were circulating in the community near the end of the testing period, the placebo patients likely acquired a serological rise in response to a wild type infection.Although we had hoped to avoid this by beginning the study in the early fall, any 'contaminating' effect of wild virus infection in the study population appeared to be small and, at any rate, would have assumedly affected each of the vaccine groups equally.
At the beginning of this study we had some difficulty in recruiting CFS patient volunteers, the most common reason for refusal being fear of some adverse effect of the vaccine on their illness.However, based on this sample, influenza vaccination has no demonstrable adverse effect on CFS.Although the patient population studied was small, and thus the chance of type 2 error large, many sequential measurements were made on the same patient, who served as his or her own control.The study was statistically sound for 'large differences', a term coined by Cohen (20) for differences that should be clinically recognizable.It is likely, although not proven, that similar results to ours would occur with other influenza vaccine antigen preparations.Thus, we feel that physicians who choose to immunize CFS patients against influenza can do so with the expectation that immunization will be effective and not have a deleterious impact on the course of the disorder.

:
CFS subjects were assigned to vaccination or placebo groups in a double-blind, randomized fashion.A computer generated program (PC PLAN, Gerard Dallal, United States), was used by the hospital pharmacy department to develop the randomization schedule.Only the pharmacist had access to the code.The investigator and all other study personnel were blinded.The pharmacy dispensed preloaded syringes labelled with each subject's initials and identification number.The research team injected CFS patients with either 0.5 mL normal saline or 0.5 mL of Fluviral S/F (IAF BioVac Inc, Canada), which is a whole virus vaccine containing antigens of A/Texas/36/91(H 1 N 1 ), A/Beijing/32/92(H 3 N 2 ) and B/Panama/45/90, recommended for the 1993-94 influenza season by the British Columbia Centre for Disease Control, Vancouver, British Columbia.Each injection was given intramuscularly into the deltoid using a 23 gauge needle, 2.5 cm in length.