Program Updates: 2004-2005

CFS Program Objectives and Strategy

The CFS program utilizes the NIH Center of Excellence strategy involving integrated activities of teams of investigators who share a common research objective–the control and prevention of CFS. The CFS research program's specific aims are to:

1. Define the magnitude and complexities of CFS as a public health problem.
2. Define the natural history and clinical parameters of CFS.
3. Identify the pathophysiology, diagnostic markers, and risk factors of CFS.
4. Educate health care professionals concerning CFS.

The program is based on a common methodology (population-based surveillance and clinical studies with a central laboratory component). The integrated CFS research program includes shared technology and other special research resources (genomics/proteomics laboratory and bioinformatics). As is pivotal to the Centers of Excellence strategy, the CFS program:

1. Brings focus and emphasis to a high priority area - CFS.
2. Integrates separate but interrelated components.
3. Provides infrastructure and shared resources.
4. Provides funding for innovative ideas.

Surveillance of CFS and other fatiguing illnesses in defined populations comprises the centerpiece of CDC's CFS research program. Initially (1989-1993), surveillance utilized sentinel physicians representing four U.S. cities–Atlanta, Grand Rapids, Reno, and Wichita. This allowed us to determine the burden imposed on the health care system by CFS, refine the case definition, evaluate the clinical course of CFS, and conduct case control studies to evaluate hypothesized infectious risk factors and immunological markers. Beginning in 1994, we shifted surveillance to defined communities. In 1994, we conducted a pilot survey of the San Francisco population. We next conducted a 4-year (1997-2001) longitudinal surveillance study of CFS and other fatiguing illnesses in the general population of Wichita. We utilized information from this study to describe the epidemiology and risk factors for CFS, explore empirical case definitions for CFS, describe its clinical course, analyze utilization of health care services by persons with CFS, estimate its economic impact, and use gene expression profiling to search for biomarkers. In order to explore unanswered questions, we conducted a pilot national survey for CFS between July 2001 and January 2002. We are currently conducting a survey of CFS in metropolitan, urban, and rural populations of Georgia.

To obtain data concerning the pathophysiology of CFS (necessary to develop control and prevention efforts), we began a series of clinical studies in 2001. Initially, these were modeling studies of CFS-like illness and included studies involving:

• immune challenge (collaborative with Emory University)
• infection (collaborative with University of New South Wales)
• exercise challenge (collaborative with National Jewish Medical Center)
• endocrine challenge (collaborative with Emory University)

In 2003, we began in-hospital studies of subjects identified with CFS from the general population. The objective of in-hospital studies is to define the pathophysiology of CFS, describe risk factors, and identify diagnostic markers. The in-hospital studies effort is unique because subjects are recruited from defined populations rather than from physician referral. Thus, results may be generalizable to the population of persons with CFS.

The genomics/proteomics/bioinformatics component of the CFS program began in 1996. It allows us to apply rapidly evolving, cutting-edge molecular biology and bioinformatics technology to epidemiologic and clinical studies. The objective of our genomics/proteomics effort is to characterize CFS at a systems biology level by applying genomics, proteomics, and other laboratory assays to specimens collected in surveillance and clinical studies and by integrating the combined data by using the most current bioinformatics tools.

The educational component of the CFS program began in the early 1990s, with telephone information lines and the development of a patient education pamphlet and a CFS Web page. In 2000, we began collaborating with the CFIDS Association of America to develop educational materials for primary care professionals. This collaboration continues, and the professional education program involves continuing medical education (CME) modules in various formats, which provide information regarding the evaluation, diagnosis, and management of CFS.

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Surveillance

Specific aims of the CFS surveillance program are to estimate the magnitude of CFS as a public health problem in the United States, describe the clinical course of CFS, devise a more sensitive and specific case definition, collect specimens to identify markers of disease activity, and identify subjects for enrollment in clinical studies.

Surveillance of CFS and Chronic Unwellness in Metropolitan, Urban, and Rural Georgia

Our studies, and those of others, have indicated that CFS may disproportionately affect minority racial/ethnic groups and those of lower income and that metropolitan and rural communities have different risks for CFS. To clarify these issues, which are central to control and prevention efforts, we began an ongoing study of CFS in metropolitan, urban, and rural populations of Georgia in May 2004. The study is using random-digit dialing to screen 10,000 residents in metropolitan (Atlanta), 10,000 residents in urban (Macon/Warner Robins), and 11,000 residents in rural (surrounding Macon/Warner Robins) counties. The study involves detailed interviews of persons reporting fatigue (~5,000) or other unwellness (~2,000) of at least a month's duration and a random sample of healthy controls (~3,000). We clinically evaluate subjects with illness resembling CFS (~300), a random sample of persons reporting other chronic unwellness (~300), and a matched set of healthy controls (~300). This one-day clinical evaluation includes assessment of subjects' medical and psychiatric status, psychosocial factors, and cognitive functioning, and a blood sample is obtained for routine testing and genotype studies. As part of the clinical evaluation, we also measure direct and indirect costs associated with CFS and assess access to and utilization of health care.

CFS Patient Registry in Metropolitan, Urban, and Rural Georgia

In 2004, CDC began planning for a CFS patient registry. The objective of the registry is to identify a large number of adults and adolescents with CFS with various durations of symptoms and to follow them over time. Specific aims of the registry are to identify well-characterized subjects for intervention trials and to describe the natural history of CFS. These specific aims require inclusion of subjects who are in the early stages of CFS (i.e., ill less than 1 year) who can be followed longitudinally to assess the course of CFS. We are currently conducting a feasibility study to assess different registry designs for efficacy and efficiency in metropolitan, urban, and rural Georgia.

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CFS Case Definition

CFS is diagnosed on the basis of self-reported symptoms—there are no pathognomonic physical signs or diagnostic laboratory tests. A sensitive and specific case definition is pivotal to public health research that seeks to design strategies to control and prevent CFS and whose efficacy can be evaluated. CDC has been the lead group internationally in developing a definition of CFS. CDC was responsible for the first published definition of CFS in 1988: an international group convened by CDC published the current international CFS research case definition in 1994. To date, definitions of CFS have represented clinical consensus rather than reflected empirical data. In 2003, CDC and the International CFS Study Group published recommendations concerning empirical application of the case definition in research studies.

Empirical Definition of CFS

The Study Group recommended studies of persons with chronic unexplained fatigue from which a definition of CFS could be empirically derived. In 2004, CDC completed analysis of two such studies. In their aggregate, they show that the core symptoms of the 1994 case definition are central to CFS. Hence, rather than changing specific criteria, we are now focusing on defining CFS on the basis of standardized, validated, and quantifiable instruments that measure the illness' major domains (disability, mental/physical fatigue, and accompanying core symptoms of the 1994 case definition). We have used this approach in clinical studies (discussed below) to clinically define CFS.

Empirical definition of CFS in Wichita

We used data collected during surveillance in Wichita to compare case-defining symptoms specified by the 1994 CFS case definition with those identified by factor analysis. We used dichotomous factor analysis to identify symptom dimensions of unexplained chronically fatiguing illness in 1,391 people. This revealed that unexplained chronic fatigue was multi-dimensional and that CFS symptoms were distributed across three factors or symptom groups: cognition-mood-sleep, inflammation, and musculoskeletal. Although a number of other symptoms were identified in each factor, those of the 1994 CFS case definition had the highest loading scores in each factor. This study was published in 2004.

Multi-center international study of patients with CFS

The aforementioned study was based on a population sample, but data were limited to residents of Wichita. We recently completed a collaborative international study to evaluate whether chronically fatiguing illness presents a common phenotype (symptom complex) across cultures. We obtained data from 37,724 patients with prolonged fatigue (n=33,753), chronic fatigue (n=2,013), or CFS (n=1,958) who were seen in 33 different studies (community, n=15,749; primary care, n=19,472; tertiary care, n=2,503). The 21 countries where data were collected included English-speaking countries (Australia, Canada, United Kingdom, Ireland, United States), non-English-speaking continental Europe (France, Germany, Greece, Italy, Spain, Sweden, The Netherlands), South America (Brazil), and non-Western cultures (Nigeria, China, Hong Kong, India, Japan, Turkey, United Arab Emirates, Vietnam). At a minimum, the data sets included:

• Demographic details and information as to the setting in which the data were collected.
• Measures of fatigue duration and severity as determined using a standard instrument such as the Chalder Fatigue Scale or Krupp Fatigue Severity Scale.
• Symptoms specified in the 1994 CFS case definition.
• Other symptoms.
• Exclusionary and non-exclusionary medical and psychiatric illnesses.

This study found symptoms segregated into four factors: perturbations of cognition, sleep disturbance, inflammation, and musculoskeletal complaints. The study also found these four factors occurred similarly in patients with unexplained fatiguing illness across cultures. A paper on this study will be submitted for publication in 2005.

Brighton Collaboration

The CFS Research Group participates actively in the Brighton Collaboration, an international group supported by CDC and WHO; the Collaboration was formed to facilitate the development, evaluation, and dissemination of high-quality information about the safety of human vaccines. The Brighton Collaboration was charged with developing a definition for evaluation of fatigue and fatiguing illnesses following immunization and formed a CFS Working Group in the summer of 2003. The Working Group, which includes participants from the United States, Canada, Brazil, The Netherlands, Argentina, Sweden, United Kingdom, and Australia, has developed a definition with three levels of diagnostic certainty for CFS following vaccination: full CFS (i.e., fatigue of at least 6 months duration, which is not relieved by rest, significantly limits usual activities, and is accompanied by at least four of the eight defining symptoms), fatigue accompanied by symptoms, and fatigue relieved by bed rest or not by at least four of the eight defining symptoms. The guidelines will be published in 2005.

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Clinical Studies

Despite more than a decade of individual investigator-initiated research, the pathophysiology of CFS remains unknown and there are no diagnostic laboratory markers and no specific treatment. This lack of success may be due to non-CFS homogeneous study groups resulting from inaccurate case ascertainment, true heterogeneity of pathophysiology within the current descriptive diagnosis of CFS, or imprecise measurement of clinical parameters. Our clinical studies effort seeks to avoid these difficulties by:

1. Enrolling patients identified from defined populations rather than referral centers.
2. Utilizing standard measures of the major symptom domains associated with CFS (as recommended above by the International CFS Study Group).
3. Conducting modeling studies of CFS-like illness following known immune, exercise, and infectious stimulus.

The clinical studies component of the CFS research program involves a multidisciplinary team of epidemiologists, physicians, mathematical statisticians, and genomics/proteomics molecular biology staff from CDC; Emory University School of Medicine faculty and support staff from the Division of Endocrinology, Department of Neurology, and Department of Psychiatry/Behavioral Medicine; and medical, laboratory, and behavioral medicine faculty at the University of New South Wales, Australia.

Clinical Assessment of Subjects with CFS and Other Fatiguing Illnesses in Wichita

Between January and July 2003, we conducted a two-day in-hospital study in Wichita with the objectives of characterizing the pathophysiology of CFS, identifying demographic, psychosocial and other risk factors, detecting biomarkers, and evaluating the CFS case definition. Assessments included:

1. Clinical evaluation of medical and psychiatric status.
2. Polysomnographic characterization of sleep characteristics.
3. Assessment of cognitive functioning.
4. Description of stress history and coping styles.
5. Laboratory studies to evaluate neuroendocrine status and autonomic nervous system function; measure cytokine levels, lymphocyte gene expression patterns, and polymorphisms in genes involved in neurotransmission and immune regulation; and describe plasma protein profiles (proteomics—SELDI-TOF).

Participants were recruited from the 659 fatigued adults clinically evaluated during the Wichita surveillance study. We attempted to enroll all those with CFS and randomly selected a similar number without CFS but with unexplained chronic fatigue (we term this ISF, insufficient symptoms of fatigue). Control subjects were randomly selected from the cohort who participated in telephone interviews at the baseline and all follow-up periods, who did not have medical or psychiatric exclusions, and who had not reported fatigue of at least 1-month duration. Each individual with CFS was matched to a control on the basis of sex, race/ethnicity, age, and body mass index. We did not match controls to the other fatigue groups because we expected to observe trends by comparing groups to each other and to the CFS subjects. Two hundred twenty-seven people participated in the study, and participation rates were similar among the recruited fatigue categories. We identified 37 people with a newly documented medical or psychiatric condition considered exclusionary for CFS, which left 190 eligible participants.

Empirical classification of CFS

We used empirical measures of functional impairment (disability), mental and physical fatigue, and CFS-defining symptoms (per recommendations of the International CFS Study Group) to diagnose CFS at the time participants came to the hospital. The Study Group recommended that rather than merely asking subjects if their fatigue caused substantial rEducation in usual activities, classification should evaluate functional impairment associated with the overall illness. We used the SF-36 health survey and defined substantial rEducation in daily activities as a score lower than the 25th percentile on the physical function, social function, role physical, or role social scales. The Study Group recommended use of a standard and validated instrument to evaluate the nature of fatigue: thus, we used the Multidimensional Fatigue Inventory (MFI) and defined severe fatigue as greater than the median of the general fatigue or reduced activity scales. Last, we used the newly validated CDC Symptom Inventory to assess the accompanying symptom complex, and we considered subjects reporting ≥4 symptoms and scoring ≥25 on the Case Definition subscale to have substantial accompanying symptoms. Defining CFS in this empirical manner increases the accuracy of case ascertainment in research studies. It also helps to clarify the extent to which patients from different referral clinics are similar (or dissimilar). This strategy can be used in primary care settings and provides health care professionals a standard and reproducible method for diagnosing CFS. This improved clinically empirical case definition for CFS was discussed with the Department of Health and Human Services CFS Advisory Committee, and a paper has been submitted for publication.

Other evaluations

The Wichita Clinical Study also assessed autonomic nervous system function, cognitive function, sleep characteristics, stress history, psychologic function, endocrine status, gene expression profiles, proteomics, and genomics. Analyses are in progress and will be published in 2005. Multivariate analyses to describe relationships between different domains (e.g., stress and sleep, stress and neuroendocrine status) will be done as a second step of analyses. We hope to begin to characterize pathophysiological pathways involved in CFS that are amenable to specific intervention.

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Modeling Studies

Investigations into the pathophysiology of CFS have focused on immune system activation secondary to infection, autoimmune reaction, and dysfunction of regulatory neuroendocrine pathways. However, these studies have been complicated by patient heterogeneity with respect to chronicity and co-morbid illnesses. In "model" systems, CFS-like illness develops following exposure to a known stimulus (or stress) obviating these problems and permitting controlled studies of the pathophysiology of fatigue and associated symptoms as they relate to immune and endocrine activation and to central nervous system response.

Post-infection fatigue - collaboration with the University of South Wales, Australia

Fatigue, cognitive disability, disturbed sleep, and myoarthralgia (i.e., the symptoms of CFS) are common during the acute phase of many infectious diseases and reflect the normal host response. Cohort studies suggest that at least 10% of individuals continue to experience these symptoms for 6-months or more after acute Q fever (Coxiella burnetii), infectious mononucleosis (Epstein-Barr virus or EBV), or epidemic polyarthritis (Ross River virus or RRV): thus, post-infectious fatigue (PIF) constitutes a subset of CFS. As with CFS, PIF is not caused by a single factor but arises from a combined action of host, microbial, and environmental factors. It is unclear how such factors interact to produce PIF/CFS, and it is also unclear whether different factors contribute to the onset, persistence, and level of disability associated with PIF/CFS. The objective of this study is to define the risk factors and pathophysiological processes that result in PIF/CFS.

We are supporting this project through a Cooperative Agreement with the University of New South Wales, Australia. The study is recruiting and following three post-infection cohorts (EBV, RRV, and Q fever). Subjects are identified and enrolled through a clinic-based active-surveillance system while they are in the acute phase of the illness (evidenced by agent-specific IgM-positive antibody response). The study uses standardized instruments to measure fatigue, psychological distress, and psychiatric morbidity over time following disease due to these three quite different agents. We also perform laboratory studies of immune response, viral and host gene expression, and proteomics (application of the techniques of molecular biology, biochemistry, and genetics for analyzing the structure, function, and interactions of the proteins produced by genes of a cell, tissue, or organism) over time. The PIF/CFS cohorts serve as model systems for identifying measurable characteristics of gene and protein expression profiles that can be correlated with immune, endocrine, and neurocognitive characteristics to differentiate and stratify the various categories of chronically fatigued from non-fatigued individuals. Andrew Lloyd, the Australian principal investigator, spent 6 months last summer on a sabbatical at CDC to work on data analysis and to prepare publications.

Two hundred fifty-three subjects have been enrolled and followed for at least 12 months. At each visit, physical and psychological health were assessed and blood samples were collected. Detailed medical, psychiatric, and laboratory evaluations were conducted at 6 months and again at 1 year to determine the occurrence of CFS. Protracted and disabling PIF/CFS, characterized by fatigue, musculo-skeletal pain, neurocognitive difficulties, and mood disturbance, occurred in 12% of subjects at 6 months and in 9% at 12 months. We specifically identified CFS in 11% of participants at 6 months. The risk of PIF/CFS was similar for all three agents, and although each of the acute infectious diseases had unique clinical features, the PIF/CFS phenotype was uniform and independent of the initial infection. PIF/CFS was predicted largely by the severity of the acute illness rather than by demographic, microbiological, immunological, or psychological factors. These infections clearly can have an etiological role in triggering CFS, and it appears that host response to infection (rather than the specific pathogen) determines the occurrence of PIF/CFS. Gene expression studies are discussed below (Genomics/Proteomics). A variety of papers exploring cytokine response and PIF/CFS are in process.

Immune system involvement in CFS' Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine

Interferon-α (IFN-α), a cytokine used in the treatment of hepatitis C (HCV) and malignant melanoma, activates the immune system and produces an illness resembling CFS (e.g., fatigue, cognitive complaints, pain, sleep disturbance, and depression). Thus, a CDC/Emory University Collaborative Group has undertaken a series of integrated studies using INF-α-associated fatigue to model CFS. Insights gained from studies of IFN-α have helped us to interpret results from the Wichita Clinical Study and to plan more targeted clinical studies of people identified in the Georgia survey for 2005.

Interferon-a-induced sickness syndrome as a model for the pathophysiology of chronic idiopathic fatigue

The cornerstone of the CDC/Emory collaboration is a study measuring immune and neuroendocrine parameters, sleep, metabolism, mood, and peripheral blood gene expression patterns in response to IFN-α therapy. Subjects coming to Emory for treatment of chronic HCV are studied prior to and after 12-weeks of IFN-α treatment. A group of patients randomized to postpone treatment until study completion serves as HCV-positive controls. The study assesses the effect of IFN-α on symptom domains relevant to CFS (fatigue, cognitive disturbance, sleep, pain, and mood/anxiety). Subjects are admitted to the Emory University General Clinical Research Center (GCRC) prior to commencing IFN-α treatment and again following 12 weeks of treatment. At both time points, they undergo assessment of fatigue (using the Checklist Individual Strength, a 20-item self-report questionnaire), objective neurocognitive assessment (using CANTAB, the Cambridge Neuropsychological Test Automated Battery, a set of tests that allows for the breakdown of complex tasks into their cognitive components), sleep polysomnography, multiple sleep latency testing, pain sensitivity testing, and medical and psychiatric assessment. Blood samples are obtained on an hourly basis from subject in the GCRC at baseline and at 12 weeks. The samples are used for assessment of cortisol, adrenocorticotropic hormone (ACTH), norepinephrine, epinephrine, cytokines (interleukin [IL]-1, IL-6, and tumor necrosis factor [TNF-α]), soluble receptors for TNF-α, and IL-6 receptors. Subjects also receive a lumbar puncture following 12-weeks of treatment with IFN-α. The lumbar puncture assesses cerebrospinal fluid concentrations of corticotropin-releasing hormone (CRH), ACTH, cortisol, norepinephrine, epinephrine, and proinflammatory cytokines (i.e., IL-1, IL-6, and TNF-α). Subjects also undergo body composition analysis, resting metabolic rate assessment, and physical activity assessment at home via actigraphy.

To date, 26 subjects (12 women, 14 men) have completed the study protocol. In the first quarter of 2005, we began analysis to examine the effect of chronic cytokine exposure on sleep and to evaluate relationships between changes in sleep architecture and the development of CFS-like symptoms. Results from these analyses will be submitted for publication by late 2005. Following sleep analyses, we will begin analyses of relationships between IFN-α-mediated changes in immune activity (including cytokine production and natural killer cell activity) and the development of CFS-like symptoms. Findings from this study will inform the design of GCRC-based immunological and neuroendocrine studies on patients with CFS identified during the Georgia Surveillance Study. CDC funding for this project has been progressively replaced by support from NIH. For example, the studies on sleep are now being supported by an NIH R01 grant. CDC funding for other IFN-α-based studies will be discontinued at the end of the current fiscal year.

Incidence of CFS in patients treated with pegylated IFN-a

Complementing the GCRC study, we have examined the incidence of fatigue and CFS in 154 patients receiving pegylated IFN-α-2b (PEG IFN) plus ribavirin for the treatment of HCV. Patients were assessed at regular intervals during the first 6 months of treatment. CFS was defined empirically by validated instruments. Fifty-five (35.7%) of patients treated with PEG IFN developed CFS during the first 6 months of treatment. Forty-nine of the remaining 99 developed fatigue that was not CFS related. These findings support the notion that chronic immune activation may participate in the pathophysiology of CFS.

Fatigue and response to viral infection

In 2004, we published data showing that the development of depressive symptoms during PEG IFN plus ribavirin treatment for HCV was associated with poor treatment response. We have extended our analysis to determine what specific symptoms accounted for this. One hundred treatment-naïve subjects with asymptomatic HCV were followed, and of the 20 items measured, only fatigue was significantly different between patients who did and did not clear virus. There was also a dose response relationship between maximum fatigue scores during treatment and viral clearing. These preliminary data suggest that fatigue during IFN-α therapy may be associated with impaired viral clearance. Likewise, fatigue in CFS may represent an altered immune response to viruses or other pathogens. Understanding the mechanisms and treatment of fatigue may yield novel strategies for improving disease outcome in CFS.

IFN-a-induced changes in cognitive function are associated with alterations in fronto-striatal circuitry

To assess whether chronic immune activation is accompanied by fronto-striatal dysfunction, we conducted a positron emission tomography (PET) study assessing changes in regional cerebral glucose metabolism in patients with malignant melanoma undergoing treatment with high doses of IFN-α. Significant decreases in glucose metabolism were apparent bilaterally in several areas of the superior frontal gyrus. Of note, increased activity in the basal ganglia, especially putamen and globus pallidus, is also seen in Parkinson's disease patients. These findings provide further support for the notion that altered function in basal ganglia circuitry, possibly related to impaired dopamine transmission, may lead to fatigue and decreased energy in patients with chronic immune activation, including patients with CFS.

Cognitive effort related to dorsal anterior cingulate cortex activation in patients treated with IFN-a

To further investigate the effects of cytokines on cognitive processing, we used functional magnetic resonance imaging (fMRI) to measure the effect of IFN-α on brain activity during a visual-spatial attention task. Twenty-one HCV patients participated in the study; 10 had received IFN-α for 12 weeks and 11 controls were awaiting IFN-α therapy. The cognitive task involved two levels of attentional demand: a low-demand, simple reaction time (detection) task and a high-demand reaction time (location) task that involved both discrimination and response selection processes. Despite evidence of fatigue and impaired concentration, IFN-α-treated patients performed the two tasks in a manner similar to controls (i.e., there were no differences in the number of errors made between groups or differences in reaction times between groups). Both IFN-α-treated patients and controls exhibited significant bilateral activation in the parietal and occipital lobes for the effect of interest (location-detection). Our data suggest that increased effort as reflected by anterior cingulate cortex (ACC) activation in IFN-α-treated patients was necessary to overcome the negative effect of fatigue on cognitive performance, especially at higher levels of task difficulty. On the basis of these findings, we anticipate that increased cognitive effort (as manifested by mental fatigue and cognitive dysfunction) in CFS patients will be associated with increased dorsal ACC activation. In addition, we hypothesize that these cortical changes will be associated with immune activation (as occurs during IFN-α treatment).

Future clinical studies

In 2005, we will concentrate on hypothesis-testing, in-hospital (GCRC) studies at Emory University of people with CFS who were identified during the Georgia survey. The objective is to identify mechanisms that translate genetic vulnerability, experience across the lifespan, and other risk factors into central nervous system, neuroendocrine, and immune dysregulation that likely underlie symptoms of CFS. The core project will characterize the physiologic and mental status of subjects representative of Georgia's metropolitan, urban, and rural populations with CFS, chronic unwellness, and other fatigue-related illnesses and will identify potential pathophysiologic mechanisms of CFS. Methods will include assessments of fatigue status, mental health status, life experiences, psychoendocrine and immune function (e.g., stress, pharmacological, and immune challenges) in order to compare them to neurochemical parameters and to results from genomic, proteomic, and genotyping studies. Structural and functional brain imaging studies (using PET, fMRI, and diffusion tensor MRI) focusing on cortical-limbic-brainstem systems will be employed to assess correlates of CFS (and its endophenotypes) at the neural systems level. To identify markers of vulnerability, study groups will comprise healthy subjects with identified risk factors for CFS.

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Genomics/Proteomics/Bioinformatics

The genomics/proteomics/bioinformatics effort integrates cutting-edge laboratory and bioinformatics methods with population, clinical, and modeling studies to characterize CFS at a systems biology level. We believe that this is the approach most likely to identify fruitful approaches to control and prevent CFS. Persons with CFS and other unexplained fatiguing illnesses are identified from defined populations, epidemiologic/clinical information, other data (as described above), and biologic specimens are collected and tested as appropriate. The genomics laboratory effort examines gene (mRNA) expression, DNA polymorphisms, and epigenetic factors; the proteomics laboratory effort aims to identify serum protein biomarkers; finally, the bioinformatics effort develops the database systems and analytical tools to integrate the data in a manner amenable to analyses. All CFS Research Program studies include genomics, proteomics, and bioinformatics, and each effort has a research and development component.

The Wichita Clinical Study

Genomics: microarray gene expression profiling

Peripheral blood samples were obtained from all participants, and peripheral blood mononuclear cells were isolated and their RNA used for microarray gene expression profiling. We have complete microarray and clinical data from 170 participants. This microarray data set, along with other clinical and epidemiologic data, will be used for C3—the CFS Computational Challenge (described below).

Genomics: DNA polymorphisms and epigenetics

The genetic contribution to risk of CFS remains largely unexplored. We are using specimens from the Wichita Clinical Study to evaluate genetic risk factors of CFS based on candidate gene and genome-wide association studies using single nucleotide polymorphism and DNA methylation assays. These studies could identify which genes, alleles, pathways, and intermediate phenotypes are at risk for CFS, and they may also identify diagnostic and therapeutic targets for CFS.

Proteomics: serum protein profiling

The genetic contribution to risk of CFS remains largely unexplored. We are using specimens from the Wichita Clinical Study to evaluate genetic risk factors of CFS based on candidate gene and genome-wide association studies using single nucleotide polymorphism and DNA methylation assays. These studies could identify which genes, alleles, pathways, and intermediate phenotypes are at risk for CFS, and they may also identify diagnostic and therapeutic targets for CFS.

We currently use surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) to obtain a semi-quantitative profile of serum proteins. Different protein spectra can be selected by using different chromatographic surfaces (protein chips). Detection is highly sensitive and generates many data end points. Complex biological samples can be analyzed and compared in a high-throughput manner to detect differentially expressed proteins in the samples. We recently completed a pilot study on serum samples from the Wichita Clinical Study to identify the optimal SELDI-TOF experimental protocol. We used 60 serum samples from CFS and non-fatigued subjects. Each sample was separated into six fractions, and each fraction was analyzed on three different protein chips. Analysis of SELDI-TOF data is in process.

Bioinformatics

Integration of data generated from the Wichita Clinical Study into models of CFS

The Wichita Clinical Study produced an enormously rich data set on the 170 participants for whom we have complete epidemiologic, clinical, and laboratory data. The data were collected to describe the various body systems thought to be disturbed in people with CFS. For example, we used standard instruments to document functional impairment in eight areas and on five dimensions of fatigue and to document the impact of accompanying symptoms; we conducted two overnight polysomnographic studies; we assessed cognitive function; we evaluated life-time stress, coping mechanisms, and other psychologic parameters in relation to levels of neuroendocrine and immune effectors in blood, saliva, and urine (i.e., hypothalamic-pituitary-adrenal [HPA] axis status). Last (as just described), we measured gene expression and protein profiles in peripheral blood. The challenge we now face is how to integrate the large volume of data into an accurate model of CFS pathogenesis.

CFS Computational Challenge

The CFS Program has organized four multidisciplinary teams to analyze epidemiologic, clinical, and gene expression data from subjects in the Wichita Clinical Study to begin to describe CFS pathogenesis. It is anticipated that each team will come up with analytical approaches and algorithms that will enable data-driven decisions for further experimentation and study design in CFS. Teams will present their solutions at a CDC-sponsored Cold Spring Harbor meeting in September 2005.

Post-Infection Fatigue: Collaborative Study with the University of South Wales

Details of the PIF (post-infectious fatigue) study were discussed above. We have completed gene expression profiling on those patients with mononucleosis, and our Australian collaborators are analyzing the data. Preliminary analysis found that expression of the MADS box transcription enhancer factor 2 polypeptide C (MEF2C) gene (involved in myogenesis) correlated with musculo-skeletal pain and fatigue, and expression of the hypocretin/orexin receptor HCRTR2 (implicated in narcolepsy) correlated with sleep dysregulation.

We are currently testing specimens collected over time from subjects with Ross River virus (RRV) and Coxiella burnetti infection (Q fever). Analysis will be approached from several angles, as these data sets can be used to examine several questions:

• Are there differences in host responses to viral and rickettsial pathogens?
• Are there differences in expression profiles between acute and recovered time points (i.e., gene expression correlates of acute illness)?
• Can gene acute-phase expression profiles predict whether a person will continue to PIF/CFS (i.e., are there differences in the acute expression profiles for subjects who resolve the infection normally compared with those who develop CFS)?

Psycho-Neuroendocrine-Immune Gene Expression and Acute Infectious Mononucleosis

Acute infection is known to perturb psycho-neuroendocrine-immune (PNI) gene expression. We used oligonucleotide microarrays to examine PNI gene expression in the peripheral blood of 13 subjects with infectious mononucleosis (IM) from the post-infective fatigue cohort. Although differential expression of PNI genes was not found in the six subjects from whom samples were obtained both during acute IM and after recovery, novel gene expression correlates of individual symptoms were discovered. Thus, investigation of the PNI response in peripheral blood may provide novel insights into the complex pathophysiology of multi-system human disease states. This work has been submitted for publication.

Acute Psychosocial Stress and Peripheral Blood Gene Expression Profiles

It is likely that CFS involves perturbations in the HPA axis. We have designed (and received a provisional patent for) a PNI oligonucleotide microarray to measure HPA axis function. We are currently evaluating endocrine, autonomic, immune, and PNI gene expression responses to stress in volunteers subjected to the Trier Social Stress Test. Blood samples are drawn at baseline (rest period), between exposure to standard stressors, and upon completion of the test. These are used for the determination of ACTH, cortisol, norepinephrine, epinephrine, and PNI gene expression studies. Our specific aims are to:

1. Validate the CDC PNI microarray, using a standardized psychosocial stress paradigm.
2. Determine the effects of acute psychosocial stress on gene expression profiles and relate molecular changes to systemic endocrine-immune responses.
3. Determine whether subjects with early-life adversity (stress-sensitive phenotype) and controls have different gene expression profiles before and after acute stress exposure.
4. Explore whether gene expression profiles after acute stress (with or without early-life stress) are similar to those previously measured in CFS patients.
5. Evaluate utility of the paradigm for CFS GCRC studies.

Research and Development

Genomics

Whole genome amplification (WGA) provides a method for increasing the amount of DNA available for use in polymorphism and epigenetic studies. DNA was subjected to genome-wide amplification, using the Genomiphi DNA amplification kit (Amersham Biosciences) to build up sufficient amounts of DNA for detection of polymorphisms by various assays on candidate genes and by genome-wide genotyping assays. DNA from all Wichita Clinical Study subjects was amplified by the WGA procedure. Pilot experiments were conducted to determine the suitability of amplified DNA for genotyping by polymerase chain reaction (PCR)-restriction fragment length polymorphism, Taqman PCR ,and pyrosequencing assays.

RNA Biorepository for CFS Biomarker Discovery and Validation

CDC's CFS program has embarked on several national and international efforts for biomarker discovery. Biorepositories of RNA from these and future studies linked to corresponding epidemiologic and clinical data are resources that are crucial to achievement these research goals. We developed a method that would make RNA in biorepositories a stable renewable resource that could be directly used in all current approaches to gene expression profiling (differential display-PCR, reverse transcription-PCR (RT-PCR), RNase protection assays, microarrays, etc.). Key features of the procedure include:

1. A modified poly-dT primer to direct reverse transcriptase to polyA transcripts and to incorporate anchoring and restriction enzyme (cloning) sites.
2. Use of the template switching activity of reverse transcriptase to incorporate a second primer with anchoring, restriction enzyme (cloning), and RNA polymerase binding sequences at the 3' end of the single-stranded first cDNA product.
3. Use of anchor-primed, limited PCR to globally copy all products to double-stranded cDNA products for archiving.

When needed, the archived material is used as template for in vitro transcription from the upstream RNA polymerase site to yield amplified RNA in the sense direction (sRNA). RT-PCR determined that sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging 5 orders of magnitude (0.00001-0.1%). This reflects the high fidelity of sRNA synthesis for mRNAs as low as 0.3-30 copies/cell. sRNA synthesis was successful with RNA from cell lines and human tissues. The double-stranded cDNA is engineered to construct cell type-specific libraries that can also serve as a stable renewable source of the original RNA. sRNA is amplified synthetic mRNA in the 5' —> 3' direction—the appropriate template for any gene expression analysis.

We have extended similar microarray studies with sRNA amplification, using a set of pilot samples from Wichita Clinical Study. Microarray data from this pilot study is under analysis to determine sensitivity and representational bias of sense RNA amplification, and to make recommendations for creation of RNA biorepositories from samples collected from past and ongoing CFS molecular epidemiologic studies.

Bioinformatics

Text mining tool

High-throughput technology, such as gene expression profiling using microarrays, allows the simultaneous assessment of tens of thousands of genes and has revolutionized our ability to piece together complex biological networks. However, utility of this glut of data is only as good as the information that can be associated with it, and several challenges complicate associating digital information from a microarray experiment with a specific gene. From a technologic point of view, proper and updated annotation of genes poses the major hurdle. From a biological perspective, genes and proteins can have multiple functions and be associated with multiple illnesses. We are developing a text mining tool that will provide relevant biological, clinical, and epidemiological information for genes identified as important by microarray gene expression profiling.

Deciphering CFS by using differential equations and multi-system object models

The HPA axis constitutes one of the major peripheral outflow systems of the brain and helps the organism adapt to changes in the external and internal environments. CRH is generated in response to physical, infectious, and psychological stress and modulates release of neurotransmitters, neuroendocrine hormones, and immune mediators that affect many bodily systems. Responses to these stressful events are manifest clinically as fatigue, impaired concentration and memory, mood changes, sleep disturbance, musculoskeletal and abdominal pain, and changes in cardiovascular function.

The sheer complexity of the numerous systems affects, the complexities of the immune and neuroendocrine systems, and the correlative nature of much of the existing data have hampered definitive research on HPA axis function in CFS. Both physical and psychological stressors activate the production and release of CRH from neurons in the hypothalamus. The CRH reaches the pituitary where it stimulates the secretion of ACTH. ACTH is released into the blood and travels to the adrenals, causing the synthesis and release of cortisol from the adrenal cortex. The release of cortisol modulates the proinflammatory cytokine response. The proinflammatory cytokines IL-1, IL-2, IL-6, and TNF-α are secreted from stimulated immune cells that signal other cells of the immune system and the hypothalamus to produce more CRH.

The objectives of this project are:

1. To construct the model by integrating multiple neuroendocrine and immune variables involved in normal HPA axis function.
2. To use the model to predict immune and neuroendocrine system dysregulation following acute physical and neurogenic stress.
3. To use the model to predict HPA axis dysregulation due to combined effects of acute and chronic stressors.

Four specific aims are involved in achieving this objective:

• Gather, review, and process existing scientific literature related to HPA axis function
• Define the objects, sub-objects, and states for use in the model
• Use the model to perform simulations
• Validate the simulations with clinical, neuroendocrine, immune function, brain imaging, gene expression (microarray), and proteomics (SELDI) data from CDC CFS research program studies.

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Education

An important component of CDC's CFS public health effort is our support of an education program for primary care professionals. The objective is to decrease morbidity associated with CFS by improving diagnosis and care of those who suffer from the illness. Specific aims are:

1. To educate the full range of primary health care professionals concerning proper evaluation, diagnosis, and care of people with CFS.
2. Introduce CFS into medical school curricula.
3. Improve public awareness and understanding of CFS.

CDC's (and other) population-based studies have estimated that about 800,000 adults in the US have CFS, most have been ill between 5 and 7 years, and a quarter of them are unemployed or receiving disability compensation. However, fewer than 20% of persons with CFS have been diagnosed and treated for the illness by a physician. This lack of appropriate care is consistent, in part, with data showing that 70% of physicians in family practice express frustration concerning appropriate management of such patients. It also reflects findings from focus groups and public surveys that indicate the public lacks sufficient basic information about CFS to seek appropriate care. Lack of a timely CFS diagnosis delays appropriate intervention and results in unnecessary anxiety, suffering, and frustration associated with failure to have a debilitating illness validated by health care professionals. Education of both health care professionals and the public about CFS is key to the effective detection, diagnosis, and care of those who suffer from CFS.

Professional Education Program

Primary Medical Care Professionals

The central aim of primary medical care professional education is to educate the full range of practicing primary health care professionals (primary care physicians, nurse practitioners, and physician assistants) about CFS in order to enable timely detection, appropriate diagnosis, and management of the illness (i.e., improved care for CFS patients). The primary professional education program began in 2000 when CDC entered into an interagency agreement with the Health Resources and Services Administration to establish a pilot CFS continuing education project conceived by the CFIDS Association of America. CDC currently manages the program under a competitively funded contract with the CFIDS Association of America. The program provides educational material and CDC-accredited CME (continuing medical education), continuing nursing education and continuing education for those who successfully completed the study. Materials are available for self-study in print, web, and video formats.

The central aim of primary medical care professional education is to educate the full range of practicing primary health care professionals (primary care physicians, nurse practitioners, and physician assistants) about CFS in order to enable timely detection, appropriate diagnosis, and management of the illness (i.e., improved care for CFS patients). The primary professional education program began in 2000 when CDC entered into an interagency agreement with the Health Resources and Services Administration to establish a pilot CFS continuing education project conceived by the CFIDS Association of America. CDC currently manages the program under a competitively funded contract with the CFIDS Association of America. The program provides educational material and CDC-accredited CME (continuing medical education), continuing nursing education and continuing education for those who successfully completed the study. Materials are available for self-study in print, web, and video formats.

Allied health professionals

In June 2003, the CFIDS Association of America convened a select group of CFS experts to shape the current medical curriculum materials into a functional format for allied health care professionals (e.g., counselors, psychologists, social workers, occupational therapists, and physical therapists). This ancillary curriculum will focus more heavily on management aspects. The allied health professional curriculum will be deployed in 2005.

Conferences

In collaboration with CDC and an advisory committee, the CFIDS Association of America has also developed a project to present the curriculum on detection, diagnosis, and management of CFS to participants at major health care professional conferences. The contract has supported development of an exhibit that advertises a self-study continuing education program, distributes print and video formats of self-study modules, provides written material on recognition, diagnosis, and management of CFS specific to each conference audience, answers questions, and conducts surveys. In addition, when possible, CFS-relevant scientific presentations are submitted to each conference and presenters are at the booth to interact with visitors.

We are currently developing formal outcome measures for the success of conference participation (e.g., booth visits, enrollment in the formal continuing education program, current attitudes/knowledge concerning CFS, and changes that follow exposure to materials). Exhibit activities and responses are monitored and discussed regularly and are continuously revised and improved. In 2003, the program exhibited at nine conferences (the American Academy of Family Physicians—attendance 6,000; the Infectious Diseases Society of America—attendance 2,500; Pri-Med East—attendance 7,500; Nurse Practitioner Associates for Continuing Education—attendance 700; American Association of CFS—attendance 700; American College of Physicians—attendance 6,500; American Academy of Physician Assistants—attendance 7,000; National Medical Association—attendance 3,500; and American Academy of Pain Management—attendance 650). In 2004, the program exhibited at 15 conferences, with a combined attendance of 28,000, and, on the average, 4% of conference attendees visited the CFS booth. Between 300 and 1,000 educational handouts were distributed at each meeting. Finally, in 2004 (the first year that complete materials were available) 435 attendees enrolled in CFS-related CME courses.

Medical School Curriculum

In addition to training practicing primary health care professionals, introduction of CFS into medical school curriculum is a major goal of the program. The CFS curriculum and targeted programs are being offered to medical schools, schools of public health, schools of nursing, and physician assistant programs and to large teaching hospitals through grand rounds/CME departments that sponsor programming for health care professionals in training and in practice. CFS experts will conduct these programs, tailoring content to reflect the clinical and research interests of each audience. Long university lead times and rigorous approval processes have delayed the launch of the first of these programs, but several institutions have expressed strong interest and have offered multiple opportunities to present to different audiences on the same campus. For example, Morehead School of Medicine would like to offer the program to its Internal Medicine, Family Practice, and Psychiatry programs. Arranging such presentations will be a major focus of effort this year, as will the constant analysis of outcomes.

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