Single-cell transcriptomics of the immune system in ME/CFS at baseline and following symptom provocation.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious and poorly understood disease. To understand immune dysregulation in ME/CFS, we use single-cell RNA sequencing (scRNA-seq) to examine immune cells in patient and control cohorts. Postexertional malaise (PEM), an exacerbation of symptoms following strenuous exercise, is a characteristic symptom of ME/CFS. To detect changes coincident with PEM, we applied scRNA-seq on the same cohorts following exercise. At baseline, ME/CFS patients display classical monocyte dysregulation suggestive of inappropriate differentiation and migration to tissue. We identify both diseased and more normal monocytes within patients, and the fraction of diseased cells correlates with disease severity. Comparing the transcriptome at baseline and postexercise challenge, we discover patterns indicative of improper platelet activation in patients, with minimal changes elsewhere in the immune system. Taken together, these data identify immunological defects present at baseline in patients and an additional layer of dysregulation in platelets.

Recovery from Exercise in Persons with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Background and Objectives: Post-exertional malaise (PEM) is the hallmark of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), but there has been little effort to quantitate the duration of PEM symptoms following a known exertional stressor. Using a Symptom Severity Scale (SSS) that includes nine common symptoms of ME/CFS, we sought to characterize the duration and severity of PEM symptoms following two cardiopulmonary exercise tests separated by 24 h (2-day CPET). Materials and Methods: Eighty persons with ME/CFS and 64 controls (CTL) underwent a 2-day CPET. ME/CFS subjects met the Canadian Clinical Criteria for diagnosis of ME/CFS; controls were healthy but not participating in regular physical activity. All subjects who met maximal effort criteria on both CPETs were included. SSS scores were obtained at baseline, immediately prior to both CPETs, the day after the second CPET, and every two days after the CPET-1 for 10 days. Results: There was a highly significant difference in judged recovery time (ME/CFS = 12.7 ± 1.2 d; CTL = 2.1 ± 0.2 d, mean ± s.e.m., Chi2 = 90.1, p < 0.0001). The range of ME/CFS patient recovery was 1-64 days, while the range in CTL was 1-10 days; one subject with ME/CFS had not recovered after one year and was not included in the analysis. Less than 10% of subjects with ME/CFS took more than three weeks to recover. There was no difference in recovery time based on the level of pre-test symptoms prior to CPET-1 (F = 1.12, p = 0.33). Mean SSS scores at baseline were significantly higher than at pre-CPET-1 (5.70 ± 0.16 vs. 4.02 ± 0.18, p < 0.0001). Pharmacokinetic models showed an extremely prolonged decay of the PEM response (Chi2 > 22, p < 0.0001) to the 2-day CPET. Conclusions: ME/CFS subjects took an average of about two weeks to recover from a 2-day CPET, whereas sedentary controls needed only two days. These data quantitate the prolonged recovery time in ME/CFS and improve the ability to obtain well-informed consent prior to doing exercise testing in persons with ME/CFS. Quantitative monitoring of PEM symptoms may provide a method to help manage PEM.

Survey of Anti-Pathogen Antibody Levels in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Infectious pathogens are implicated in the etiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) because of the occurrence of outbreaks of the disease. While a number of different infectious agents have been associated with the onset of ME/CFS, the identity of a specific organism has been difficult to determine in individual cases. The aim of our study is to survey ME/CFS subjects for evidence of an infectious trigger and/or evidence of immune dysregulation via serological testing of plasma samples for antibodies to 122 different pathogen antigens. Immune profiles were compared to age-, sex-, and BMI-matched controls to provide a basis for comparison. Antibody levels to individual antigens surveyed in this study do not implicate any one of the pathogens in ME/CFS, nor do they rule out common pathogens that frequently infect the US population. However, our results revealed sex-based differences in steady-state humoral immunity, both within the ME/CFS cohort and when compared to trends seen in the healthy control cohort.

Plasma metabolomics reveals disrupted response and recovery following maximal exercise in myalgic encephalomyelitis/chronic fatigue syndrome.

Post-exertional malaise (PEM) is a hallmark symptom of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We monitored the evolution of 1157 plasma metabolites in 60 ME/CFS (45 female, 15 male) and 45 matched healthy control participants (30 female, 15 male) before and after 2 maximal cardiopulmonary exercise test (CPET) challenges separated by 24 hours, with the intent of provoking PEM in patients. Four time points allowed exploration of the metabolic response to maximal energy-producing capacity and the recovery pattern of participants with ME/CFS compared with the healthy control group. Baseline comparison identified several significantly different metabolites, along with an enriched percentage of yet-to-be identified compounds. Additionally, temporal measures demonstrated an increased metabolic disparity between cohorts, including unknown metabolites. The effects of exertion in the ME/CFS cohort predominantly highlighted lipid-related as well as energy-related pathways and chemical structure clusters, which were disparately affected by the first and second exercise sessions. The 24-hour recovery period was distinct in the ME/CFS cohort, with over a quarter of the identified pathways statistically different from the controls. The pathways that are uniquely different 24 hours after an exercise challenge provide clues to metabolic disruptions that lead to PEM. Numerous altered pathways were observed to depend on glutamate metabolism, a crucial component of the homeostasis of many organs in the body, including the brain.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Essentials of Diagnosis and Management.

Despite myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) affecting millions of people worldwide, many clinicians lack the knowledge to appropriately diagnose or manage ME/CFS. Unfortunately, clinical guidance has been scarce, obsolete, or potentially harmful. Consequently, up to 91% of patients in the United States remain undiagnosed, and those diagnosed often receive inappropriate treatment. These problems are of increasing importance because after acute COVID-19, a significant percentage of people remain ill for many months with an illness similar to ME/CFS. In 2015, the US National Academy of Medicine published new evidence-based clinical diagnostic criteria that have been adopted by the US Centers for Disease Control and Prevention. Furthermore, the United States and other governments as well as major health care organizations have recently withdrawn graded exercise and cognitive-behavioral therapy as the treatment of choice for patients with ME/CFS. Recently, 21 clinicians specializing in ME/CFS convened to discuss best clinical practices for adults affected by ME/CFS. This article summarizes their top recommendations for generalist and specialist health care providers based on recent scientific progress and decades of clinical experience. There are many steps that clinicians can take to improve the health, function, and quality of life of those with ME/CFS, including those in whom ME/CFS develops after COVID-19. Patients with a lingering illness that follows acute COVID-19 who do not fully meet criteria for ME/CFS may also benefit from these approaches.

In-Depth Analysis of the Plasma Proteome in ME/CFS Exposes Disrupted Ephrin-Eph and Immune System Signaling.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disabling disease with worldwide prevalence and limited therapies exclusively aimed at treating symptoms. To gain insights into the molecular disruptions in ME/CFS, we utilized an aptamer-based technology that quantified 4790 unique human proteins, allowing us to obtain the largest proteomics dataset yet available for this disease, detecting highly abundant proteins as well as rare proteins over a nine-log dynamic range. We report a pilot study of 20 ME/CFS patients and 20 controls, all females. Significant differences in the levels of 19 proteins between cohorts implicate pathways related to the extracellular matrix, the immune system and cell-cell communication. Outputs of pathway and cluster analyses robustly highlight the ephrin pathway, which is involved in cell-cell signaling and regulation of an expansive variety of biological processes, including axon guidance, angiogenesis, epithelial cell migration, and immune response. Receiver Operating Characteristic (ROC) curve analyses distinguish the plasma proteomes of ME/CFS patients from controls with a high degree of accuracy (Area Under the Curve (AUC) > 0.85), and even higher when using protein ratios (AUC up to 0.95), that include some protein pairs with established biological relevance. Our results illustrate the promise of plasma proteomics for diagnosing and deciphering the molecular basis of ME/CFS.

Cytokine profiling of extracellular vesicles isolated from plasma in myalgic encephalomyelitis/chronic fatigue syndrome: a pilot study.

BACKGROUND: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease of unknown etiology lasting for a minimum of 6 months but usually for many years, with features including fatigue, cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Dysregulation of cytokine signaling could give rise to many of these symptoms. Cytokines are present in both plasma and extracellular vesicles, but little investigation of EVs in ME/CFS has been reported. Therefore, we aimed to characterize the content of extracellular vesicles (EVs) isolated from plasma (including circulating cytokine/chemokine profiling) from individuals with ME/CFS and healthy controls. METHODS: We included 35 ME/CFS patients and 35 controls matched for age, sex and BMI. EVs were enriched from plasma by using a polymer-based precipitation method and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM) and immunoblotting. A 45-plex immunoassay was used to determine cytokine levels in both plasma and isolated EVs from a subset of 19 patients and controls. Linear regression, principal component analysis and inter-cytokine correlations were analyzed. RESULTS: ME/CFS individuals had significantly higher levels of EVs that ranged from 30 to 130 nm in size as compared to controls, but the mean size for total extracellular vesicles did not differ between groups. The enrichment of typical EV markers CD63, CD81, TSG101 and HSP70 was confirmed by Western blot analysis and the morphology assessed by TEM showed a homogeneous population of vesicles in both groups. Comparison of cytokine concentrations in plasma and isolated EVs of cases and controls yielded no significant differences. Cytokine-cytokine correlations in plasma revealed a significant higher number of interactions in ME/CFS cases along with 13 inverse correlations that were mainly driven by the Interferon gamma-induced protein 10 (IP-10), whereas in the plasma of controls, no inverse relationships were found across any of the cytokines. Network analysis in EVs from controls showed 2.5 times more significant inter-cytokine interactions than in the ME/CFS group, and both groups presented a unique negative association. CONCLUSIONS: Elevated levels of 30-130 nm EVs were found in plasma from ME/CFS patients and inter-cytokine correlations revealed unusual regulatory relationships among cytokines in the ME/CFS group that were different from the control group in both plasma and EVs. These disturbances in cytokine networks are further evidence of immune dysregulation in ME/CFS.

Comprehensive Circulatory Metabolomics in ME/CFS Reveals Disrupted Metabolism of Acyl Lipids and Steroids.

The latest worldwide prevalence rate projects that over 65 million patients suffer from myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), an illness with known effects on the functioning of the immune and nervous systems. We performed an extensive metabolomics analysis on the plasma of 52 female subjects, equally sampled between controls and ME/CFS patients, which delivered data for about 1750 blood compounds spanning 20 super-pathways, subdivided into 113 sub-pathways. Statistical analysis combined with pathway enrichment analysis points to a few disrupted metabolic pathways containing many unexplored compounds. The most intriguing finding concerns acyl cholines, belonging to the fatty acid metabolism sub-pathway of lipids, for which all compounds are consistently reduced in two distinct ME/CFS patient cohorts. We compiled the extremely limited knowledge about these compounds and regard them as promising in the quest to explain many of the ME/CFS symptoms. Another class of lipids with far-reaching activity on virtually all organ systems are steroids; androgenic, progestin, and corticosteroids are broadly reduced in our patient cohort. We also report on lower dipeptides and elevated sphingolipids abundance in patients compared to controls. Disturbances in the metabolism of many of these molecules can be linked to the profound organ system symptoms endured by ME/CFS patients.

Prospective Biomarkers from Plasma Metabolomics of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Implicate Redox Imbalance in Disease Symptomatology.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a disease of enigmatic origin with no established cure. Its constellation of symptoms has silently ruined the lives of millions of people around the world. A plethora of hypotheses have been vainly investigated over the past few decades, so that the biological basis of this debilitating condition remains a mystery. In this study, we investigate whether there is a disturbance in homeostasis of metabolic networks in the plasma of a female 32-patient cohort compared to 19 healthy female controls. Extensive analysis of the 832-metabolite dataset generated by Metabolon®, covering eight biological classes, generated important insight into metabolic disruptions that occur in ME/CFS. We report on 14 metabolites with differences in abundance, allowing us to develop a theory of broad redox imbalance in ME/CFS patients, which is consistent with findings of prior work in the ME/CFS field. Moreover, exploration of enrichment analysis using www.MetaboAnalyst.ca provides information concerning similarities between metabolite disruptions in ME/CFS and those that occur in other diseases, while its biomarker analysis unit yielded prospective plasma biomarkers for ME/CFS. This work contributes key elements to the development of ME/CFS diagnostics, a crucial step required for discovering a therapy for any disease of unknown origin.

Eukaryotes in the gut microbiota in myalgic encephalomyelitis/chronic fatigue syndrome.

Patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) often suffer from gastrointestinal symptoms and many are diagnosed with irritable bowel syndrome (IBS). Previous studies, including from our laboratory, have demonstrated that the ME/CFS gut bacterial composition is altered and less diverse when compared to healthy individuals. Patients have increased biomarkers of inflammation and leaky gut syndrome. To further investigate dysbiosis in the ME/CFS gut microbiome, we sought to characterize the eukaryotes present in the gut of 49 individuals with ME/CFS and 39 healthy controls. Using 18S rRNA sequencing, we have identified eukaryotes in stool samples of 17 healthy individuals and 17 ME/CFS patients. Our analysis demonstrates a small, nonsignificant decrease in eukaryotic diversity in ME/CFS patients compared to healthy individuals. In addition, ME/CFS patients show a nonsignificant increase in the ratio of fungal phyla Basidiomycota to Ascomycota, which is consistent with ongoing inflammation in ME/CFS. We did not identify specific eukaryotic taxa that are associated with ME/CFS disease status.

Metabolic profiling of a myalgic encephalomyelitis/chronic fatigue syndrome discovery cohort reveals disturbances in fatty acid and lipid metabolism.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) remains a continuum spectrum disease without biomarkers or simple objective tests, and therefore relies on a diagnosis from a set of symptoms to link the assortment of brain and body disorders to ME/CFS. Although recent studies show various affected pathways, the underlying basis of ME/CFS has yet to be established. In this pilot study, we compare plasma metabolic signatures in a discovery cohort, 17 patients and 15 matched controls, and explore potential metabolic perturbations as the aftermath of the complex interactions between genes, transcripts and proteins. This approach to examine the complex array of symptoms and underlying foundation of ME/CFS revealed 74 differentially accumulating metabolites, out of 361 (P < 0.05), and 35 significantly altered after statistical correction (Q < 0.15). The latter list includes several essential energy-related compounds which could theoretically be linked to the general lack of energy observed in ME/CFS patients. Pathway analysis points to a few pathways with high impact and therefore potential disturbances in patients, mainly taurine metabolism and glycerophospholipid metabolism, combined with primary bile acid metabolism, as well as glyoxylate and dicarboxylate metabolism and a few other pathways, all involved broadly in fatty acid metabolism. Purines, including ADP and ATP, pyrimidines and several amino acid metabolic pathways were found to be significantly disturbed. Finally, glucose and oxaloacetate were two main metabolites affected that have a major effect on sugar and energy levels. Our work provides a prospective path for diagnosis and understanding of the underlying mechanisms of ME/CFS.

Reduced diversity and altered composition of the gut microbiome in individuals with myalgic encephalomyelitis/chronic fatigue syndrome.

BACKGROUND: Gastrointestinal disturbances are among symptoms commonly reported by individuals diagnosed with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). However, whether ME/CFS is associated with an altered microbiome has remained uncertain. Here, we profiled gut microbial diversity by sequencing 16S ribosomal ribonucleic acid (rRNA) genes from stool as well as inflammatory markers from serum for cases (n = 48) and controls (n = 39). We also examined a set of inflammatory markers in blood: C-reactive protein (CRP), intestinal fatty acid-binding protein (I-FABP), lipopolysaccharide (LPS), LPS-binding protein (LBP), and soluble CD14 (sCD14). RESULTS: We observed elevated levels of some blood markers for microbial translocation in ME/CFS patients; levels of LPS, LBP, and sCD14 were elevated in ME/CFS subjects. Levels of LBP correlated with LPS and sCD14 and LPS levels correlated with sCD14. Through deep sequencing of bacterial rRNA markers, we identified differences between the gut microbiomes of healthy individuals and patients with ME/CFS. We observed that bacterial diversity was decreased in the ME/CFS specimens compared to controls, in particular, a reduction in the relative abundance and diversity of members belonging to the Firmicutes phylum. In the patient cohort, we find less diversity as well as increases in specific species often reported to be pro-inflammatory species and reduction in species frequently described as anti-inflammatory. Using a machine learning approach trained on the data obtained from 16S rRNA and inflammatory markers, individuals were classified correctly as ME/CFS with a cross-validation accuracy of 82.93 %. CONCLUSIONS: Our results indicate dysbiosis of the gut microbiota in this disease and further suggest an increased incidence of microbial translocation, which may play a role in inflammatory symptoms in ME/CFS.

Increased ventricular lactate in chronic fatigue syndrome measured by 1H MRS imaging at 3.0 T. II: comparison with major depressive disorder.

Chronic fatigue syndrome (CFS), a complex illness characterized by fatigue, impaired concentration, and musculoskeletal pain, is often misdiagnosed as a psychiatric illness due to the overlap of its symptoms with mood and anxiety disorders. Using proton magnetic resonance spectroscopic imaging ((1)H MRSI), we previously measured levels of the major brain metabolites in CFS, in generalized anxiety disorder (GAD), and in healthy control subjects, and found significantly higher levels of ventricular cerebrospinal fluid (CSF) lactate in CFS compared to the other two groups. In the present study, we sought to assess the specificity of this observation for CFS by comparing ventricular lactate levels in a new cohort of 17 CFS subjects with those in 19 healthy volunteers and in 21 subjects with major depressive disorder (MDD), which, like GAD, is a neuropsychiatric disorder that has significant symptom overlap with CFS. Ventricular CSF lactate was significantly elevated in CFS compared to healthy volunteers, replicating the major result of our previous study. Ventricular lactate measures in MDD did not differ from those in either CFS or healthy volunteers. We found a significant correlation between ventricular CSF lactate and severity of mental fatigue that was specific to the CFS group. In an exploratory analysis, we did not find evidence for altered levels of the amino acid neurotransmitters, gamma-aminobutyric acid (GABA) and glutamate + glutamine ('Glx'), in CFS compared to MDD or healthy controls. Future (1)H MRS studies with larger sample sizes and well-characterized populations will be necessary to further clarify the sensitivity and specificity of neurometabolic abnormalities in CFS and MDD.

Sensitive, qualitative detection of human herpesvirus-6 and simultaneous differentiation of variants A and B.

BACKGROUND: The current limitations of laboratory testing for the detection of human herpesvirus virus 6 (HHV-6) in clinical specimens with low HHV-6 viral loads make this area a priority for further research and development. OBJECTIVES: To develop and validate a sensitive qualitative assay for simultaneous HHV-6 detection and variant differentiation. METHODS: We developed a diagnostic procedure, which combines a magnetic bead-based nucleic acid extraction, PCR amplification, and colorimetric microtiter plate identification (MAG-PCR-EIA), for the sensitive detection of HHV-6 and the simultaneous differentiation of HHV-6A and HHV-6B. RESULTS: Analytic sensitivities of the MAG-PCR-EIA assay were 10 copies per reaction for both HHV-6A and HHV-6B variants, which is equivalent to 20 copies/ml when 1ml of clinical specimen was processed. A proficiency panel containing 11 blinded specimens covering HHV-6A viral loads from 0 to 100,000 copies was tested, and the MAG-PCR-EIA was able to detect the lowest concentration at one copy in 200microl. A panel of 27 urine specimens, which were collected from patients with and without chronic fatigue syndrome, was tested by the MAG-PCR-EIA. HHV-6 was detected in two (HHV-6A) patients who have chromosomally integrated HHV-6A and in one (HHV-6B) patient who was a healthy control and diagnosed as cervical cancer later on. The HHV-6 results did not correlate with results previously determined by HHV-6 antigenemia in urine. CONCLUSION: With large specimen volumes processed and an additional signal amplification incorporated, the MAG-PCR-EIA provides a sensitive and qualitative system for HHV-6 detection and simultaneous variant differentiation. Clinical relevance of the assay awaits further investigation.

Ventricular cerebrospinal fluid lactate is increased in chronic fatigue syndrome compared with generalized anxiety disorder: an in vivo 3.0 T (1)H MRS imaging study.

Chronic fatigue syndrome (CFS) is a controversial diagnosis because of the lack of biomarkers for the illness and its symptom overlap with neuropsychiatric, infectious, and rheumatological disorders. We compared lateral ventricular volumes derived from tissue-segmented T(1)-weighted volumetric MRI data and cerebrospinal fluid (CSF) lactate concentrations measured by proton MRS imaging ((1)H MRSI) in 16 subjects with CFS (modified US Centers for Disease Control and Prevention criteria) with those in 14 patients with generalized anxiety disorder (GAD) and in 15 healthy volunteers, matched group-wise for age, sex, body mass index, handedness, and IQ. Mean lateral ventricular lactate concentrations measured by (1)H MRSI in CFS were increased by 297% compared with those in GAD (P < 0.001) and by 348% compared with those in healthy volunteers (P < 0.001), even after controlling for ventricular volume, which did not differ significantly between the groups. Regression analysis revealed that diagnosis accounted for 43% of the variance in ventricular lactate. CFS is associated with significantly raised concentrations of ventricular lactate, potentially consistent with recent evidence of decreased cortical blood flow, secondary mitochondrial dysfunction, and/or oxidative stress abnormalities in the disorder.

Chronic fatigue syndrome: what role does the autonomic nervous system play in the pathophysiology of this complex illness?

Chronic fatigue syndrome (CFS) is a serious health concern affecting over 800000 Americans of all ages, races and socioeconomic groups and both genders. The etiology and pathophysiology of CFS are unknown, yet studies have suggested an involvement of the autonomic nervous system (ANS). A symposium was organized in December 2000 to explore the possibility of an association between ANS dysfunction and CFS, with special emphasis on the interactions between ANS dysfunction and other abnormalities noted in the immune and endocrine systems of individuals with CFS. This paper represents the consensus of the panel of experts who participated in this meeting.